Amino-Derivatives As Novel Inhibitors Of Histone Deacetylase

ABSTRACT

This invention comprises the novel compounds of formula (I) 
     
       
         
         
             
             
         
       
     
     wherein n, m, t, R 1 , R 2 , L, Q, X, Y, Z and 
     
       
         
         
             
             
         
       
     
     have defined meanings, having histone deacetylase inhibiting enzymatic activity; their preparation, compositions containing them and their use as a medicine.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/429,838, filed Apr. 24, 2009, which is a divisional of U.S.application Ser. No. 10/507,784, now U.S. Pat. No. 7,541,369, filed Sep.13, 2004, which is the National State Entry of International ApplicationNo. PCT/EP2003/002513, filed Mar. 11, 2003, which claims the benefit ofU.S. Provisional Application No. 60/363,799, filed Mar. 13, 2002.

This invention concerns compounds having histone deacetylase (HDAC)inhibiting enzymatic activity. It further relates to processes for theirpreparation, to compositions comprising them, as well as their use, bothin vitro and in vivo, to inhibit HDAC and as a medicine, for instance asa medicine to inhibit proliferative conditions, such as cancer andpsoriasis.

In all eukaryotic cells, genomic DNA in chromatine associates withhistones to form nucleosomes. Each nucleosome consists of a proteinoctamer made up of two copies of each histones H2A, H2B, H3 and H4. DNAwinds around this protein core, with the basic amino acids of thehistones interacting with the negatively charged phosphate groups of theDNA. The most common posttranslational modification of these corehistones is the reversible acetylation of the c-amino groups ofconserved, highly basic N-terminal lysine residues. The steady state ofhistone acetylation is established by the dynamic equilibrium betweencompeting histone acetyltransferase(s) and histone deacetylase(s) hereinreferred to as “HDAC”. Histone acetylation and deacetylation has longbeen linked to transcriptional control. The recent cloning of the genesencoding different histone acetyltransferases and histone deacetylasesprovided a possible explanation for the relationship between histoneacetylation and transcriptional control. The reversible acetylation ofhistones can result in chromatin remodelling and as such act as acontrol mechanism for gene transcription. In general, hyperacetylationof histones facilitates gene expression, whereas histone deacetylationis correlated with transcriptional repression. Histoneacetyltransferases were shown to act as transcriptional coactivators,whereas histone deacetylases were found to belong to transcriptionalrepression pathways.

The dynamic equilibrium between histone acetylation and deacetylation isessential for normal cell growth. Inhibition of histone deacetylaseresults in cell cycle arrest, cellular differentiation, apoptosis andreversal of the transformed phenotype. Therefore HDAC inhibitors canhave great therapeutic potential in the treatment of cell proliferativediseases or conditions (Marks et al., Nature Reviews: Cancer 1: 194-202,2001)

The study of inhibitors of histone deacetylases (HDAC) indicates thatindeed these enzymes play an important role in cell proliferation anddifferentiation. The inhibitor Trichostatin A (TSA) causes cell cyclearrest at both G1 and G2 phases, reverts the transformed phenotype ofdifferent cell lines, and induces differentiation of Friend leukemiacells and others. TSA (and suberoylanilide hydroxamic acid SAHA) havebeen reported to inhibit cell growth, induce terminal differentiation,and prevent the formation of tumours in mice (Finnin et al., Nature,401: 188-193, 1999).

Trichostatin A has also been reported to be useful in the treatment offibrosis, e.g. liver fibrosis and liver chirrhosis. (Geerts et al.,European Patent Application EP 0 827 742, published 11 Mar., 1998).

Patent application DE 4228792 published on Mar. 3, 1994 describescompounds of general formula

It provides processes for the preparation of these compounds,compositions comprising them, as well as their use as a fungicide.

Patent application WO 97/23216 published on Jul. 3, 1997 describesselective antagonists of N-methyl-D-aspartate receptor subtypes ofgeneral formula

It presents pharmaceutical compositions containing these compounds anddiscloses the usefulness of these compounds for the treatment ofneurological and other conditions.

U.S. Pat. No. 5,952,349 published on Sep. 14, 1999 discloses compoundsof general formula

It further describes pharmaceutical compositions containing saidcompounds, the use of said compounds for the treatment of cognitivedisorders and the use of said compounds in combination withacetylcholinesterase inhibitors.

Patent application WO 00/71516 published on Nov. 30, 2000 describescompounds of general formula A-Y-D-E-G-J-Z-L having activity againstmammalian factor Xa, pharmaceutical compositions containing them and theuse of said compounds for preventing or treating coagulation disorders.

Patent application WO 02/42271 describes compounds of general formula

It further provides pharmaceutical compositions comprising thesecompounds as well as the use of these compounds as a medicine for thetreatment of hyperlipidemia, obesity and type II diabetes.

Patent application WO 02/00651 published on Jan. 3, 2002 disclosesinhibitors of trypsin-like serine protease enzymes of general formulas

It presents pharmaceutical compositions containing said compounds andthe use of said compounds as anti-coagulant agents for treatment andprevention of thromboembolic disorders.

Patent application WO 02/18335 published on Mar. 7, 2002 disclosescompounds of general formula

It provides for the use of said compounds in the treatment ofCCR3-related diseases.

Patent application WO 03/000653 published on 3 Jan., 2003 describescompounds of general formula

It further provides processes for the preparation of these compounds,compositions comprising them, as well as their use as athrombin-inhibiting agent.

The compounds of the present invention differ from the prior artdescribed in DE4228792, WO 97/23216, U.S. Pat. No. 5,952,349, WO00/71516, WO 02/00651, WO 02/18335 and WO 03/000653 in structure and inactivity.

Patent application WO01/38322 published on May 31, 2001 disclosesamongst others inhibitors of histone deacetylase of general formulaCy-L¹-Ar—Y¹—C(O)—NH—Z, providing compositions and methods for treatingcell proliferative diseases and conditions.

Patent application WO01/70675 published on 27 Sep., 2001 disclosesinhibitors of histone deacetylase of formula Cy²-Cy¹-X—Y¹—W andCy-S(O)₂—NH—Y³—W and further provides compositions and methods fortreating cell proliferative diseases and conditions.

The problem to be solved is to provide histone deacetylase inhibitorswith high enzymatic activity and also show advantageous properties suchas cellular activity and increased bioavailability, preferably oralbioavailability, and have little or no side effects.

The novel compounds of the present invention solve the above describedproblem. The compounds differ from the prior art in structure.

The compounds of the present invention show excellent in-vitro histonedeacetylase inhibiting enzymatic activity. The present compounds haveadvantageous properties with regard to cellular activity and specificproperties with regard to inhibition of cell cycle progression at bothG1 and G2 checkpoints (p21 induction capacity). The compounds of thepresent invention show good metabolic stability and high bioavailabilityand more particular they show oral bioavailability.

This invention concerns compounds of formula (I)

the N-oxide forms, the pharmaceutically acceptable addition salts andthe stereo-chemically isomeric forms thereof, wherein

-   n is 0, 1, 2 or 3 and when n is 0 then a direct bond is intended;-   m is 0, 1, 2 or 3 and when m is 0 then a direct bond is intended; 10-   t is 0 or 1 and when t is 0 then a direct bond is intended;-   each Q is nitrogen or

-   each X is nitrogen or

-   each Y is nitrogen or

-   each Z is —CH₂— or —O—;-   R¹ is —C(O)NR³R⁴, —N(H)C(O)R², —C(O)—C₁₋₆alkanediylSR⁷,    —NR⁸C(O)N(OH)R⁷, —NR⁸C(O)C₁₋₆alkanediylSR⁷, —NR⁸C(O)C═N(OH)R² or    another Zn-chelating-group wherein R³ and R⁴ are each independently    selected from hydrogen, hydroxy, C₁₋₆alkyl, hydroxyC₁₋₆alkyl,    aminoC₁₋₆alkyl or aminoaryl; R⁷ is independently selected from    hydrogen, C₁₋₆alkyl, C₁₋₆alkylcarbonyl, arylC₁₋₆alkyl,    C₁₋₆alkylpyrazinyl, pyridinone, pyrrolidinone or methylimidazolyl;    R⁸ is independently selected from hydrogen or C₁₋₆alkyl;-   R² is hydrogen, hydroxy, amino, hydroxyC₁₋₆alkyl, C₁₋₆alkyl,    C₁₋₆alkyloxy, arylC₁₋₆alkyl, aminocarbonyl, hydroxycarbonyl,    aminoC₁₋₆alkyl, aminocarbonylC₁₋₆alkyl, hydroxycarbonylC₁₋₆alkyl,    hydroxyaminocarbonyl, C₁₋₆alkyloxycarbonyl, C₁₋₆alkylaminoC₁₋₆alkyl    or di(C₁₋₆allyl)aminoC₁₋₆alkyl;-   -L- is a bivalent radical selected from C₁₋₆alkanediyl, carbonyl,    sulfonyl, or C₁₋₆alkanediyl substituted with phenyl;

is a radical selected from

-   wherein each s is independently 0, 1, 2, 3, 4 or 5;-   each R⁵ and R⁶ are independently selected from hydrogen; halo;    hydroxy; amino; nitro; trihaloC₁₋₆alkyl; trihaloC₁₋₆alkyloxy;    C₁₋₆alkyl; C₁₋₆alkyl substituted with aryl and C₃₋₁₀cycloalkyl;    C₁₋₆alkyloxy; C₁₋₆alkyloxyC₁₋₆alkyloxy; C₁₋₆alkylcarbonyl;    C₁₋₆alkyloxycarbonyl; C₁₋₆alkylsulfonyl; cyanoC₁₋₆alkyl;    hydroxyC₁₋₆alkyl; hydroxyC₁₋₆alkyloxy; hydroxyC₁₋₆alkylamino;    aminoC₁₋₆alkyloxy; di(C₁₋₆alkyl)aminocarbonyl;    di(hydroxyC₁₋₆alkyl)amino; (aryl)(C₁₋₆alkyl)amino;    di(C₁₋₆alkyl)aminoC₁₋₆alkyloxy; di(C₁₋₆alkyl)aminoC₁₋₆alkylamino;    di(C₁₋₆alkyl)aminoC₁₋₆alkylaminoC₁₋₆alkyl; arylsulfonyl;    arylsulfonylamino; aryloxy; aryloxyC₁₋₆alkyl; arylC₂₋₆alkenediyl;    di(C₁₋₆alkyl)amino; di(C₁₋₆alkyl)aminoC₁₋₆alkyl;    di(C₁₋₆alkyl)amino(C₁₋₆alkyl)amino;    di(C₁₋₆alkyl)amino(C₁₋₆alkyl)aminoC₁₋₆alkyl;    di(C₁₋₆alkyl)aminoC₁₋₆alkyl(C₁₋₆alkyl)amino;    di(C₁₋₆alkyl)aminoC₁₋₆alkyl(C₁₋₆alkyl)aminoC₁₋₆alkyl;    aminosulfonylamino(C₁₋₆alkyl)amino; amino sulfonylamino    (C₁₋₆alkyl)aminoC₁₋₆alkyl;    di(C₁₋₆alkyl)aminosulfonylamino(C₁₋₆alkyl)amino;    di(C₁₋₆alkyl)aminosulfonylamino(C₁₋₆alkyl)aminoC₁₋₆alkyl; cyano;    thiophenyl; thiophenyl substituted with    di(C₁₋₆alkyl)aminoC₁₋₆alkyl(C₁₋₆alkyl)aminoC₁₋₆alkyl,    di(C₁₋₆alkyl)aminoC₁₋₆alkyl, C₁₋₆alkylpiperazinylC₁₋₆alkyl,    hydroxyC₁₋₆alkylpiperazinylC₁₋₆alkyl,    hydroxyC₁₋₆alkyloxyC₁₋₆alkylpiperazinylC₁₋₆alkyl,    di(C₁₋₆alkyl)aminosulfonylpiperazinylC₁₋₆alkyl,    C₁₋₆alkyloxypiperidinyl, C₁₋₆alkyloxypiperidinylC₁₋₆alkyl,    morpholinylC₁₋₆alkyl, hydroxyC₁₋₆alkyl(C₁₋₆alkyl)aminoC₁₋₆alkyl, or    di(hydroxyC₁₋₆alkyl)aminoC₁₋₆alkyl; furanyl; furanyl substituted    with hydroxyC₁₋₆alkyl; benzofuranyl; imidazolyl; oxazolyl; oxazolyl    substituted with aryl and C₁₋₆alkyl; C₁₋₆alkyltriazolyl; tetrazolyl;    pyrrolidinyl; pyrrolyl; piperidinylC₁₋₆alkyloxy; morpholinyl;    C₁₋₆alkylmorpholinyl; morpholinylC₁₋₆alkyloxy; morpholinylC₁₋₆alkyl;    morpholinylC₁₋₆alkylamino; morpholinylC₁₋₆alkylaminoC₁₋₆alkyl;    piperazinyl; C₁₋₆alkylpiperazinyl; C₁₋₆alkylpiperazinylC₁₋₆alkyloxy;    piperazinylC₁₋₆alkyl; naphtalenylsulfonylpiperazinyl;    naphtalenylsulfonylpiperidinyl; naphtalenylsulfonyl;    C₁₋₆alkylpiperazinylC₁₋₆alkyl; C₁₋₆alkylpiperazinylC₁₋₆alkylamino;    C₁₋₆alkylpiperazinylC₁₋₆alkylaminoC₁₋₆alkyl;    C₁₋₆alkylpiperazinylsulfonyl; aminosulfonylpiperazinylC₁₋₆alkyloxy;    aminosulfonylpiperazinyl; aminosulfonylpiperazinylC₁₋₆alkyl;    di(C₁₋₆alkyl)aminosulfonylpiperazinyl;    di(C₁₋₆alkyl)aminosulfonylpiperazinylC₁₋₆alkyl;    hydroxyC₁₋₆alkylpiperazinyl; hydroxyC₁₋₆alkylpiperazinylC₁₋₆alkyl;    C₁₋₆alkyloxypiperidinyl; C₁₋₆alkyloxypiperidinylC₁₋₆alkyl;    piperidinylaminoC₁₋₆alkylamino;    piperidinylaminoC₁₋₆alkylaminoC₁₋₆alkyl;    (C₁₋₆alkylpiperidinyl)(hydroxyC₁₋₆alkyl)aminoC₁₋₆alkylamino;    (C₁₋₆alkylpiperidinyl)(hydroxyC₁₋₆alkyl)aminoC₁₋₆alkylaminoC₁₋₆alkyl;    hydroxyC₁₋₆alkyloxyC₁₋₆alkylpiperazinyl;    hydroxyC₁₋₆alkyloxyC₁₋₆alkylpiperazinylC₁₋₆alkyl;    (hydroxyC₁₋₆alkyl)(C₁₋₆alkyl)amino;    (hydroxyC₁₋₆alkyl)(C₁₋₆alkyl)aminoC₁₋₆alkyl;    hydroxyC₁₋₆alkylaminoC₁₋₆alkyl; di(hydroxyC₁₋₆alkyl)aminoC₁₋₆alkyl;    pyrrolidinylC₁₋₆alkyl; pyrrolidinylC₁₋₆alkyloxy; pyrazolyl;    thiopyrazolyl; pyrazolyl substituted with two substituents selected    from C₁₋₆alkyl or trihaloC₁₋₆alkyl; pyridinyl; pyridinyl substituted    with C₁₋₆alkyloxy, aryloxy or aryl; pyrimidinyl;    tetrahydropyrimidinylpiperazinyl;    tetrahydropyrimidinylpiperazinylC₁₋₆alkyl; quinolinyl; indolyl;    phenyl; phenyl substituted with one, two or three substituents    independently selected from halo, amino, nitro, C₁₋₆alkyl,    C₁₋₆alkyloxy, hydroxyC₁₋₄alkyl, trifluoromethyl, trifluoromethyloxy,    hydroxyC₁₋₄alkyloxy, C₁₋₄alkylsulfonyl, C₁₋₄alkyloxyC₁₋₄alkyloxy,    C₁₋₄alkyloxycarbonyl, aminoC₁₋₄alkyloxy,    di(C₁₋₄alkyl)aminoC₁₋₄alkyloxy, di(C₁₋₄alkyl)amino,    di(C₁₋₄alkyl)aminocarbonyl, di(C₁₋₄alkyl)aminoC₁₋₄alkyl,    di(C₁₋₄alkyl)aminoC₁₋₄alkylaminoC₁₋₄alkyl,    di(C₁₋₄alkyl)amino(C₁₋₄alkyl)amino,    di(C₁₋₄alkyl)amino(C₁₋₄alkyl)aminoC₁₋₄alkyl,    di(C₁₋₄alkyl)aminoC₁₋₄alkyl(C₁₋₄alkyl)amino,    di(C₁₋₄alkyl)aminoC₁₋₄alkyl(C₁₋₄alkyl)aminoC₁₋₄alkyl,    aminosulfonylamino(C₁₋₄alkyl)amino, amino sulfonylamino    (C₁₋₄alkyl)aminoC₁₋₄alkyl,    di(C₁₋₄alkyl)aminosulfonylamino(C₁₋₄alkyl)amino,    di(C₁₋₄alkyl)aminosulfonylamino(C₁₋₄alkyl)aminoC₁₋₆alkyl, cyano,    piperidinylC₁₋₄alkyloxy, pyrrolidinylC₁₋₄alkyloxy,    aminosulfonylpiperazinyl, aminosulfonylpiperazinylC₁₋₄alkyl,    di(C₁₋₄alkyl)aminosulfonylpiperazinyl,    di(C₁₋₄alkyl)aminosulfonylpiperazinylC₁₋₄alkyl,    hydroxyC₁₋₄alkylpiperazinyl, hydroxyC₁₋₄alkylpiperazinylC₁₋₄alkyl,    C₁₋₄alkyloxypiperidinyl, C₁₋₄alkyloxypiperidinylC₁₋₄alkyl,    hydroxyC₁₋₄alkyloxyC₁₋₄alkylpiperazinyl,    hydroxyC₁₋₄alkyloxyC₁₋₄alkylpiperazinylC₁₋₄alkyl,    (hydroxyC₁₋₄alkyl)(C₁₋₄alkyl)amino,    (hydroxyC₁₋₄alkyl)(C₁₋₄alkyl)aminoC₁₋₄alkyl,    di(hydroxyC₁₋₄alkyl)amino, di(hydroxyC₁₋₄alkyl)aminoC₁₋₄alkyl,    furanyl, furanyl substituted with —CH═CH—CH═CH—,    pyrrolidinylC₁₋₄alkyl, pyrrolidinylC₁₋₄alkyloxy, morpholinyl,    morpholinylC₁₋₄alkyloxy, morpholinylC₁₋₄alkyl,    morpholinylC₁₋₄alkylamino, morpholinylC₁₋₄alkylaminoC₁₋₄alkyl,    piperazinyl, C₁₋₄alkylpiperazinyl, C₁₋₄alkylpiperazinylC₁₋₄alkyloxy,    piperazinylC₁₋₄alkyl, C₁₋₄alkylpiperazinylC₁₋₄alkyl,    C₁₋₄alkylpiperazinylC₁₋₄alkylamino,    C₁₋₄alkylpiperazinylC₁₋₄alkylaminoC₁₋₆alkyl,    tetrahydropyrimidinylpiperazinyl,    tetrahydropyrimidinylpiperazinylC₁₋₄alkyl,    piperidinylaminoC₁₋₄alkylamino,    piperidinylaminoC₁₋₄alkylaminoC₁₋₄alkyl,    (C₁₋₄alkylpiperidinyl)(hydroxyC₁₋₄alkyl)aminoC₁₋₄alkylamino,    (C₁₋₄alkylpiperidinyl)(hydroxyC₁₋₄alkyl)aminoC₁₋₄alkylaminoC₁₋₄alkyl,    pyridinylC₁₋₄alkyloxy, hydroxyC₁₋₄alkylamino,    hydroxyC₁₋₄alkylaminoC₁₋₄alkyl, di(C₁₋₄alkyl)aminoC₁₋₄alkylamino,    aminothiadiazolyl, aminosulfonylpiperazinylC₁₋₄alkyloxy, or    thiophenylC₁₋₄alkylamino;-   each R⁵ and R⁶ can be placed on the nitrogen in replacement of the    hydrogen;-   aryl in the above is phenyl, or phenyl substituted with one or more    substituents each independently selected from halo, C₁₋₆alkyl,    C₁₋₆alkyloxy, trifluoromethyl, cyano or hydroxycarbonyl.

The term “histone deacetylase inhibitor” or “inhibitor of histonedeacetylase” is used to identify a compound, which is capable ofinteracting with a histone deacetylase and inhibiting its activity, moreparticularly its enzymatic activity. Inhibiting histone deacetylaseenzymatic activity means reducing the ability of a histone deacetylaseto remove an acetyl group from a histone. Preferably, such inhibition isspecific, i.e. the histone deacetylase inhibitor reduces the ability ofa histone deacetylase to remove an acetyl group from a histone at aconcentration that is lower than the concentration of the inhibitor thatis required to produce some other, unrelated biological effect.

As used in the foregoing definitions and hereinafter, halo is generic tofluoro, chloro, bromo and iodo; C₁₋₄alkyl defines straight and branchedchain saturated hydrocarbon radicals having from 1 to 4 carbon atomssuch as, e.g. methyl, ethyl, propyl, butyl, 1-methylethyl,2-methylpropyl and the like; C₁₋₆alkyl includes C₁₋₄alkyl and the higherhomologues thereof having 5 to 6 carbon atoms such as, for example,pentyl, 2-methyl-butyl, hexyl, 2-methylpentyl and the like;C₁₋₆alkanediyl defines bivalent straight and branched chained saturatedhydrocarbon radicals having from 1 to 6 carbon atoms such as, forexample, methylene, 1,2-ethanediyl, 1,3-propanediyl 1,4-butanediyl,1,5-pentanediyl, 1,6-hexanediyl and the branched isomers thereof suchas, 2-methylpentanediyl, 3-methylpentanediyl, 2,2-dimethylbutanediyl,2,3-dimethylbutanediyl and the like; trihaloC₁₋₆alkyl defines C₁₋₆alkylcontaining three identical or different halo substituents for exampletrifluoromethyl; C₂₋₆alkenediyl defines bivalent straight and branchedchain hydrocarbon radicals containing one double bond and having from 2to 6 carbon atoms such as, for example, ethenediyl, 2-propenediyl,3-butenediyl, 2-pentenediyl, 3-pentenediyl, 3-methyl-2-butenediyl, andthe like; and aminoaryl defines aryl substituted with amino.

The term “another Zn-chelating group” refers to a group, which iscapable of interacting with a Zn-ion, which can be present at anenzymatic binding site.

Pharmaceutically acceptable addition salts encompass pharmaceuticallyacceptable acid addition salts and pharmaceutically acceptable baseaddition salts. The pharmaceutically acceptable acid addition salts asmentioned hereinabove are meant to comprise the therapeutically activenon-toxic acid addition salt forms, which the compounds of formula (I)are able to form. The compounds of formula (I) which have basicproperties can be converted in their pharmaceutically acceptable acidaddition salts by treating said base form with an appropriate acid.Appropriate acids comprise, for example, inorganic acids such ashydrohalic acids, e.g. hydrochloric or hydrobromic acid; sulfuric;nitric; phosphoric and the like acids; or organic acids such as, forexample, acetic, trifluoroacetic, propanoic, hydroxyacetic, lactic,pyruvic, oxalic, malonic, succinic (i.e. butanedioic acid), maleic,fumaric, malic, tartaric, citric, methanesulfonic, ethanesulfonic,benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic,p-amino-salicylic, pamoic and the like acids.

The compounds of formula (I) which have acidic properties may beconverted in their pharmaceutically acceptable base addition salts bytreating said acid form with a suitable organic or inorganic base.Appropriate base salt forms comprise, for example, the ammonium salts,the alkali and earth alkaline metal salts, e.g. the lithium, sodium,potassium, magnesium, calcium salts and the like, salts with organicbases, e.g. the benzathine, N-methyl-D-glucamine, hydrabamine salts, andsalts with amino acids such as, for example, arginine, lysine and thelike.

The term “acid or base addition salts” also comprises the hydrates andthe solvent addition forms, which the compounds of formula (I) are ableto form. Examples of such forms are e.g. hydrates, alcoholates and thelike.

The term “stereochemically isomeric forms of compounds of formula (I)”,as used herein, defines all possible compounds made up of the same atomsbonded by the same sequence of bonds but having differentthree-dimensional structures, which are not interchangeable, which thecompounds of formula (I) may possess. Unless otherwise mentioned orindicated, the chemical designation of a compound encompasses themixture of all possible stereochemically isomeric forms, which saidcompound might possess. Said mixture may contain all diastereomersand/or enantiomers of the basic molecular structure of said compound.All stereochemically isomeric forms of the compounds of formula (I) bothin pure form or in admixture with each other are intended to be embracedwithin the scope of the present invention.

The N-oxide forms of the compounds of formula (I) are meant to comprisethose compounds of formula (I) wherein one or several nitrogen atoms areoxidized to the so-called N-oxide, particularly those N-oxides whereinone or more of the piperidine-, piperazine or pyridazinyl-nitrogens areN-oxidized.

Some of the compounds of formula (I) may also exist in their tautomericforms. Such forms although not explicitly indicated in the above formulaare intended to be included within the scope of the present invention.

Whenever used hereinafter, the term “compounds of formula (I)” is meantto include also the pharmaceutically acceptable addition salts and allstereoisomeric forms.

As used herein, the terms “histone deacetylase” and “HDAC” are intendedto refer to any one of a family of enzymes that remove acetyl groupsfrom the c-amino groups of lysine residues at the N-terminus of ahistone. Unless otherwise indicated by context, the term “histone” ismeant to refer to any histone protein, including H1, H2A, H2B, H3, H4,and H5, from any species. Human HDAC proteins or gene products, include,but are not limited to, HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-6,HDAC-7, HDAC-8, HDAC-9 and HDAC-10. The histone deacetylase can also bederived from a protozoal or fungal source.

A first group of interesting compounds consists of those compounds offormula (I) wherein one or more of the following restrictions apply:

-   a) n is 0, 1 or 2;-   b) m is 0, 1 or 2;-   c) each Q is i

-   d) each X is nitrogen;-   e) R¹ is —C(O)NH(OH);-   f) R² is hydrogen;-   g)-L- is a bivalent radical selected from carbonyl, sulfonyl, or    C₁₋₆alkanediyl substituted with phenyl;-   h)

is a radical selected from (a-1), (a-20) or (a-43);

-   i) each s is independently 0 or 1;-   j) each R⁵ is independently selected from hydrogen or phenyl.

A second group of interesting compounds consists of those compounds offormula (I) wherein one or more of the following restrictions apply:

a) n is 0, 1 or 2;

b) m is 1 or 2;

c) each Q isd) each X is nitrogen;

d) R¹ is —C(O)NH(OH);

f) R² is hydrogen;e) -L- is a bivalent radical selected from carbonyl or sulfonyl;

-   f)

is a radical selected from (a-1) or (a-20);

-   g) each s is independently 0 or 1;-   h) each R⁵ is independently selected from hydrogen or aryl.

A third group of interesting compounds consists of those compounds offormula (I) wherein R¹ is —C(O)NH(OH).

A fourth group of interesting compounds consists of those compounds offormula (I) wherein one or more of the following restrictions apply:

-   a) t is 0;-   b) R¹ is —C(O)NR³R⁴, —C(O)—C₁₋₆alkanediylSR⁷, —NR⁸C(O)N(OH)R⁷,    —NR⁸C(O)C₁₋₆alkanediylSR⁷, —NR⁸C(O)C═N(OH)R⁷ or another    Zn-chelating-group wherein R³ and R⁴ are each independently selected    from hydrogen, hydroxy, hydroxyC₁₋₆alkyl or aminoC₁₋₆alkyl;-   c) R² is hydrogen, hydroxy, amino, hydroxyC₁₋₆alkyl, C₁₋₆alkyl,    C₁₋₆alkyloxy, arylC₁₋₆alkyl, aminocarbonyl, aminoC₁₋₆alkyl,    C₁₋₆alkylaminoC₁₋₆alkyl or di(C₁₋₆alkyl)aminoC₁₋₆alkyl;-   d) -L- is a bivalent radical selected from C₁₋₆alkanediyl, carbonyl    or sulfonyl;-   e)

is a radical selected from (a-1), (a-3), (a-4), (a-5), (a-6), (a-7),(a-8), (a-9), (a-10), (a-11), (a-12), (a-13), (a-14), (a-15), (a-16),(a-17), (a-18), (a-19), (a-20), (a-21), (a-22), (a-23), (a-24), (a-25),(a-26), (a-28), (a-29), (a-30), (a-31), (a-32), (a-33), (a-34), (a-35),(a-36), (a-37), (a-38), (a-39), (a-40), (a-41), (a-42), (a-44), (a-45),(a-46), (a-47), (a-48) or (a-51);

-   f) each s is independently 0, 1, 2, 3 or 4;-   g) R⁵ is hydrogen; halo; hydroxy; amino; nitro; trihaloC₁₋₆alkyl;    trihaloC₁₋₆alkyloxy; C₁₋₆alkyl; C₁₋₆alkyloxy; C₁₋₆alkylcarbonyl;    C₁₋₆alkyloxycarbonyl; C₁₋₆alkylsulfonyl; hydroxyC₁₋₆ alkyl; aryloxy;    di(C₁₋₆alkyl)amino; cyano; thiophenyl; furanyl; furanyl substituted    with hydroxyC₁₋₆alkyl; benzofuranyl; imidazolyl; oxazolyl; oxazolyl    substituted with aryl and C₁₋₆alkyl; C₁₋₆alkyltriazolyl; tetrazolyl;    pyrrolidinyl; pyrrolyl; morpholinyl; C₁₋₆alkylmorpholinyl;    piperazinyl; C₁₋₆alkylpiperazinyl; hydroxyC₁₋₆alkylpiperazinyl;    C₁₋₆alkyloxypiperidinyl; pyrazoly; pyrazolyl substituted with one or    two substituents selected from C₁₋₆alkyl or trihaloC₁₋₆alkyl;    pyridinyl; pyridinyl substituted with C₁₋₆alkyloxy, aryloxy or aryl;    pyrimidinyl; quinolinyl; indole; phenyl; or phenyl substituted with    one or two substituents independently selected from halo, C₁₋₆alkyl,    C₁₋₆alkyloxy or trifluoromethyl;-   h) R⁶ is hydrogen; halo; hydroxy; amino; nitro; trihaloC₁₋₆alkyl;    trihaloC₁₋₆alkyloxy; C₁₋₆alkyl; C₁₋₆alkyloxy; C₁₋₆alkylcarbonyl;    C₁₋₆alkyloxycarbonyl; C₁₋₆alkylsulfonyl; hydroxyC₁₋₆alkyl; aryloxy;    di(C₁₋₆alkyl)amino; cyano; pyridinyl; phenyl; or phenyl substituted    with one or two substituents independently selected from halo,    C₁₋₆alkyl, C₁₋₆alkyloxy or trifluoromethyl.

A group of preferred compounds consists of those compounds of formula(I) wherein

-   t is 0;-   R¹ is —C(O)NR³R⁴, —C(O)—C₁₋₆alkanediylSR², —NR⁸C(O)N(OH)R⁷,    —NR⁸C(O)C₁₋₆alkanediylSR², —NR⁸C(O)C═N(OH)R⁷ or another    Zn-chelating-group wherein R³ and R⁴ are each independently selected    from hydrogen, hydroxy, hydroxyC₁₋₆alkyl or aminoC₁₋₆alkyl;-   R² is hydrogen, hydroxy, amino, hydroxyC₁₋₆alkyl, C₁₋₆alkyl,    C₁₋₆alkyloxy, arylC₁₋₆alkyl, aminocarbonyl, aminoC₁₋₆alkyl,    C₁₋₆alkylaminoC₁₋₆alkyl or di(C₁₋₆alkyl)aminoC₁₋₆alkyl;-   -L- is a bivalent radical selected from C₁₋₆alkanediyl, carbonyl or    sulfonyl;

is a radical selected from (a-1), (a-3), (a-4), (a-5), (a-6), (a-7),(a-8), (a-9), (a-10), (a-11), (a-12), (a-13), (a-14), (a-15), (a-16),(a-17), (a-18), (a-19), (a-20), (a-21), (a-22), (a-23), (a-24), (a-25),(a-26), (a-28), (a-29), (a-30), (a-31), (a-32), (a-33), (a-34), (a-35),(a-36), (a-37), (a-38), (a-39), (a-40), (a-41), (a-42), (a-44), (a-45),(a-46), (a-47), (a-48) or (a-51);

-   s is 0, 1, 2, 3 or 4;-   R⁵ is hydrogen; halo; hydroxy; amino; nitro; trihaloC₁₋₆alkyl;    trihaloC₁₋₆alkyloxy; C₁₋₆alkyl; C₁₋₆alkyloxy; C₁₋₆ alkylcarbonyl;    C₁₋₆ alkyloxycarbonyl; C₁₋₆alkylsulfonyl; hydroxyC₁₋₆alkyl; aryloxy;    di(C₁₋₆alkyl)amino; cyano; thiophenyl; furanyl; furanyl substituted    with hydroxyC₁₋₆alkyl; benzofuranyl; imidazolyl; oxazolyl; oxazolyl    substituted with aryl and C₁₋₆alkyl; C₁₋₆alkyltriazolyl; tetrazolyl;    pyrrolidinyl; pyrrolyl; morpholinyl; C₁₋₆alkylmorpholinyl;    piperazinyl; C₁₋₆alkylpiperazinyl; hydroxyC₁₋₆alkylpiperazinyl;    C₁₋₆alkyloxypiperidinyl; pyrazoly; pyrazolyl substituted with one or    two substituents selected from C₁₋₆alkyl or trihaloC₁₋₆alkyl;    pyridinyl; pyridinyl substituted with C₁₋₆alkyloxy, aryloxy or aryl;    pyrimidinyl; quinolinyl; indole; phenyl; or phenyl substituted with    one or two substituents independently selected from halo, C₁₋₆alkyl,    C₁₋₆alkyloxy or trifluoromethyl; and-   R⁶ is hydrogen; halo; hydroxy; amino; nitro; trihaloC₁₋₆alkyl;    trihaloC₁₋₆alkyloxy; C₁₋₆alkyl; C₁₋₆alkyloxy; C₁₋₆alkylcarbonyl;    C₁₋₆alkyloxycarbonyl; C₁₋₆alkylsulfonyl; hydroxyC₁₋₆alkyl; aryloxy;    di(C₁₋₆alkyl)amino; cyano; pyridinyl; phenyl; or phenyl substituted    with one or two substituents independently selected from halo,    C₁₋₆alkyl, C₁₋₆alkyloxy or trifluoromethyl.

A group of more preferred compounds consists of those compounds offormula (I) wherein n is 0, 1 or 2; m is 0, 1 or 2; each Q is

each X is nitrogen; R¹ is —C(O)NH(OH); R² is hydrogen; -L- is a bivalentradical selected from carbonyl, sulfonyl, or C₁₋₆alkanediyl substitutedwith phenyl;

is a radical selected from (a-1), (a-20) or (a-43); s is 0 or 1; andeach R⁵ is independently selected from hydrogen or phenyl.

A group of even more preferred compounds consists of those compounds offormula (I) wherein n is 0, 1 or 2; m is 1 or 2; each Q is

each X is nitrogen; R¹ is —C(O)NH(OH); R² is hydrogen; -L- is a bivalentradical selected from carbonyl or sulfonyl;

is a radical selected from (a-1) or (a-20); each s is independently 0 or1; and each R⁵ is independently selected from hydrogen or aryl.

Most preferred compounds are compounds No. 1, No. 3, No. 5, No. 4, No.14, No. 12, No. 7, No. 8 or No. 9.

The compounds of formula (I) and their pharmaceutically acceptable saltsand N-oxides and stereochemically isomeric forms thereof may be preparedin conventional manner.

A general synthesis route is encompassed as example:

a) Hydroxamic acids of formula (I) wherein R¹ is —C(O)NH(OH), saidcompounds being referred to as compounds of formula (I-a), may beprepared by reacting an intermediate of formula (II) with an appropriateacid, such as for example, trifluoro acetic acid. Said reaction isperformed in an appropriate solvent, such as, for example, methanol.

b) intermediates of formula (II) may be prepared by reacting anintermediate of formula (III) with an intermediate of formula (IV) inthe presence of appropriate reagents such asN′-(ethylcarbonimidoyl)-N,N-dimethyl-1,3-propanediamine,monohydrochloride (EDC) and 1-hydroxy-1H-benzotriazole (HOBT). Thereaction may be performed in a suitable solvent such as a mixture ofdichloromethane and tetrahydrofuran.

c) intermediates of formula (III) may be prepared by reacting anintermediate of formula (V) with an appropriate base such as NaOH in thepresence of a suitable solvent such as ethanol.

The compounds of formula (I) can also conveniently be prepared usingsolid phase synthesis techniques. In general, solid phase synthesisinvolves reacting an intermediate in a synthesis with a polymer support.This polymer-supported intermediate can then be carried on through anumber of synthesis steps. After each step, filtering the resin andwashing it numerous times with various solvents remove impurities. Ateach step the resin can be split up to react with various intermediatesin the next step thus allowing for the synthesis of a large number ofcompounds. After the last step in the procedure the resin is treatedwith a reagent or process to cleave the resin from the sample. Moredetailed explanation of the techniques used in solid phase chemistry isdescribed in for example “The Combinatorial Index” (B. Bunin, AcademicPress) and Novabiochem's 1999 Catalogue & Peptide Synthesis Handbook(Novabiochem AG, Switzerland) both incorporated herein by reference.

The compounds of formula (I) and some of the intermediates may have atleast one stereogenic centre in their structure. This stereogenic centremay be present in an R or an S configuration.

The compounds of formula (I) as prepared in the hereinabove describedprocesses are generally racemic mixtures of enantiomers, which can beseparated from one another following art-known resolution procedures.The racemic compounds of formula (I) may be converted into thecorresponding diastereomeric salt forms by reaction with a suitablechiral acid. Said diastereomeric salt forms are subsequently separated,for example, by selective or fractional crystallization and theenantiomers are liberated there from by alkali. An alternative manner ofseparating the enantiomeric forms of the compounds of formula (I)involves liquid chromatography using a chiral stationary phase. Saidpure stereochemically isomeric forms may also be derived from thecorresponding pure stereochemically isomeric forms of the appropriatestarting materials, provided that the reaction occursstereospecifically. Preferably if a specific stereoisomer is desired,said compound would be synthesized by stereospecific methods ofpreparation. These methods will advantageously employ enantiomericallypure starting materials.

The compounds of formula (I), the pharmaceutically acceptable acidaddition salts and stereoisomeric forms thereof have valuablepharmacological properties in that they have a histone deacetylase(HDAC) inhibitory effect.

This invention provides a method for inhibiting the abnormal growth ofcells, including transformed cells, by administering an effective amountof a compound of the invention. Abnormal growth of cells refers to cellgrowth independent of normal regulatory mechanisms (e.g. loss of contactinhibition). This includes the inhibition of tumour growth both directlyby causing growth arrest, terminal differentiation and/or apoptosis ofcancer cells, and indirectly, by inhibiting neovascularization oftumours.

This invention also provides a method for inhibiting tumour growth byadministering an effective amount of a compound of the presentinvention, to a subject, e.g. a mammal (and more particularly a human)in need of such treatment. In particular, this invention provides amethod for inhibiting the growth of tumours by the administration of aneffective amount of the compounds of the present invention. Examples oftumours which may be inhibited, but are not limited to, lung cancer(e.g. adenocarcinoma and including non-small cell lung cancer),pancreatic cancers (e.g. pancreatic carcinoma such as, for exampleexocrine pancreatic carcinoma), colon cancers (e.g. colorectalcarcinomas, such as, for example, colon adenocarcinoma and colonadenoma), prostate cancer including the advanced disease, hematopoietictumours of lymphoid lineage (e.g. acute lymphocytic leukemia, B-celllymphoma, Burkitt's lymphoma), myeloid leukemias (for example, acutemyelogenous leukemia (AML)), thyroid follicular cancer, myelodysplasticsyndrome (MDS), tumours of mesenchymal origin (e.g. fibrosarcomas andrhabdomyosarcomas), melanomas, teratocarcinomas, neuroblastomas,gliomas, benign tumour of the skin (e.g. keratoacanthomas), breastcarcinoma (e.g. advanced breast cancer), kidney carcinoma, ovarycarcinoma, bladder carcinoma and epidermal carcinoma.

The compound according to the invention may be used for othertherapeutic purposes, for example:

-   -   a) the sensitisation of tumours to radiotherapy by administering        the compound according to the invention before, during or after        irradiation of the tumour for treating cancer;    -   b) treating arthropathies and osteopathological conditions such        as rheumatoid arthritis, osteoarthritis, juvenile arthritis,        gout, polyarthritis, psoriatic arthritis, ankylosing spondylitis        and systemic lupus erythematosus;    -   c) inhibiting smooth muscle cell proliferation including        vascular proliferative disorders, atherosclerosis and        restenosis;    -   d) treating inflammatory conditions and dermal conditions such        as ulcerative colitis, Crohn's disease, allergic rhinitis, graft        vs. host disease, conjunctivitis, asthma, ARDS, Behcets disease,        transplant rejection, uticaria, allergic dermatitis, alopecia        areata, scleroderma, exanthema, eczema, dermatomyositis, acne,        diabetes, systemic lupus erythematosis, Kawasaki's disease,        multiple sclerosis, emphysema, cystic fibrosis and chronic        bronchitis;    -   e) treating endometriosis, uterine fibroids, dysfunctional        uterine bleeding and endometrial hyperplasia;    -   f) treating ocular vascularisation including vasculopathy        affecting retinal and choroidal vessels;    -   g) treating a cardiac dysfunction;    -   h) inhibiting immunosuppressive conditions such as the treatment        of HIV infections;    -   i) treating renal dysfunction;    -   j) suppressing endocrine disorders;    -   k) inhibiting dysfunction of gluconeogenesis;    -   l) treating a neuropathology for example Parkinson's disease or        a neuropathology that results in a cognitive disorder, for        example, Alzheimer's disease or polyglutamine related neuronal        diseases;    -   m) inhibiting a neuromuscular pathology, for example,        amylotrophic lateral sclerosis;    -   n) treating spinal muscular atrophy;    -   o) treating other pathologic conditions amenable to treatment by        potentiating expression of a gene;    -   p) enhancing gene therapy.

Hence, the present invention discloses the compounds of formula (I) foruse as a medicine as well as the use of these compounds of formula (I)for the manufacture of a medicament for treating one or more of theabove mentioned conditions.

The compounds of formula (I), the pharmaceutically acceptable acidaddition salts and stereoisomeric forms thereof can have valuablediagnostic properties in that they can be used for detecting oridentifying a HDAC in a biological sample comprising detecting ormeasuring the formation of a complex between a labelled compound and aHDAC.

The detecting or identifying methods can use compounds that are labelledwith labelling agents such as radioisotopes, enzymes, fluorescentsubstances, luminous substances, etc. Examples of the radioisotopesinclude ¹²⁵I, ¹³¹I, ³H and ¹⁴C. Enzymes are usually made detectable byconjugation of an appropriate substrate which, in turn catalyses adetectable reaction. Examples thereof include, for example,beta-galactosidase, beta-glucosidase, alkaline phosphatase, peroxidaseand malate dehydrogenase, preferably horseradish peroxidase. Theluminous substances include, for example, luminol, luminol derivatives,luciferin, aequorin and luciferase.

Biological samples can be defined as body tissue or body fluids.Examples of body fluids are cerebrospinal fluid, blood, plasma, serum,urine, sputum, saliva and the like.

In view of their useful pharmacological properties, the subjectcompounds may be formulated into various pharmaceutical forms foradministration purposes.

To prepare the pharmaceutical compositions of this invention, aneffective amount of a particular compound, in base or acid addition saltform, as the active ingredient is combined in intimate admixture with apharmaceutically acceptable carrier, which carrier may take a widevariety of forms depending on the form of preparation desired foradministration. These pharmaceutical compositions are desirably inunitary dosage form suitable, preferably, for administration orally,rectally, percutaneously, or by parenteral injection. For example, inpreparing the compositions in oral dosage form, any of the usualpharmaceutical media may be employed, such as, for example, water,glycols, oils, alcohols and the like in the case of oral liquidpreparations such as suspensions, syrups, elixirs and solutions; orsolid carriers such as starches, sugars, kaolin, lubricants, binders,disintegrating agents and the like in the case of powders, pills,capsules and tablets.

Because of their ease in administration, tablets and capsules representthe most advantageous oral dosage unit form, in which case solidpharmaceutical carriers are obviously employed. For parenteralcompositions, the carrier will usually comprise sterile water, at leastin large part, though other ingredients, to aid solubility for example,may be included. Injectable solutions, for example, may be prepared inwhich the carrier comprises saline solution, glucose solution or amixture of saline and glucose solution. Injectable suspensions may alsobe prepared in which case appropriate liquid carriers, suspending agentsand the like may be employed. In the compositions suitable forpercutaneous administration, the carrier optionally comprises apenetration enhancing agent and/or a suitable wetting agent, optionallycombined with suitable additives of any nature in minor proportions,which additives do not cause a significant deleterious effect to theskin. Said additives may facilitate the administration to the skinand/or may be helpful for preparing the desired compositions. Thesecompositions may be administered in various ways, e.g., as a transdermalpatch, as a spot-on or as an ointment.

It is especially advantageous to formulate the aforementionedpharmaceutical compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used in thespecification and claims herein refers to physically discrete unitssuitable as unitary dosages, each unit containing a predeterminedquantity of active ingredient, calculated to produce the desiredtherapeutic effect, in association with the required pharmaceuticalcarrier. Examples of such dosage unit forms are tablets (includingscored or coated tablets), capsules, pills, powder packets, wafers,injectable solutions or suspensions, teaspoonfuls, tablespoonfuls andthe like, and segregated multiples thereof.

Those skilled in the art could easily determine the effective amountfrom the test results presented hereinafter. In general it iscontemplated that a therapeutically effective amount would be from 0.05mg/kg to 100 mg/kg body weight, and in particular from 0.05 mg/kg to 10mg/kg body weight. It may be appropriate to administer the required doseas two, three, four or more sub-doses at appropriate intervalsthroughout the day. Said sub-doses may be formulated as unit dosageforms, for example, containing 0.5 to 500 mg, and in particular 10 mg to500 mg of active ingredient per unit dosage form.

As another aspect of the present invention a combination of aHDAC-inhibitor with another anticancer agent is envisaged, especiallyfor use as a medicine, more specifically in the treatment of cancer orrelated diseases.

For the treatment of the above conditions, the compounds of theinvention may be advantageously employed in combination with one or moreother medicinal agents, more particularly, with other anti-canceragents. Examples of anti-cancer agents are:

-   -   platinum coordination compounds for example cisplatin,        carboplatin or oxalyplatin;    -   taxane compounds for example paclitaxel or docetaxel;    -   topoisomerase I inhibitors such as camptothecin compounds for        example irinotecan or topotecan;    -   topoisomerase II inhibitors such as anti-tumour podophyllotoxin        derivatives for example etoposide or teniposide;    -   anti-tumour vinca alkaloids for example vinblastine, vincristine        or vinorelbine;    -   anti-tumour nucleoside derivatives for example 5-fluorouracil,        gemcitabine or capecitabine;    -   alkylating agents such as nitrogen mustard or nitrosourea for        example cyclophosphamide, chlorambucil, carmustine or lomustine;    -   anti-tumour anthracycline derivatives for example daunorubicin,        doxorubicin, idarubicin or mitoxantrone;    -   HER2 antibodies for example trastuzumab;    -   estrogen receptor antagonists or selective estrogen receptor        modulators for example tamoxifen, toremifene, droloxifene,        faslodex or raloxifene;    -   aromatase inhibitors such as exemestane, anastrozole, letrazole        and vorozole;    -   differentiating agents such as retinoids, vitamin D and retinoic        acid metabolism blocking agents (RAMBA) for example accutane;    -   DNA methyl transferase inhibitors for example azacytidine;    -   kinase inhibitors for example flavoperidol, imatinib mesylate or        gefitinib;    -   farnesyltransferase inhibitors; or    -   other HDAC inhibitors.

The term “platinum coordination compound” is used herein to denote anytumor cell growth inhibiting platinum coordination compound whichprovides platinum in the form of an ion.

The term “taxane compounds” indicates a class of compounds having thetaxane ring system and related to or derived from extracts from certainspecies of yew (Taxus) trees.

The term “topisomerase inhibitors” is used to indicate enzymes that arecapable of altering DNA topology in eukaryotic cells. They are criticalfor important cellular functions and cell proliferation. There are twoclasses of topoisomerases in eukaryotic cells, namely type I and typeII. Topoisomerase I is a monomeric enzyme of approximately 100,000molecular weight. The enzyme binds to DNA and introduces a transientsingle-strand break, unwinds the double helix (or allows it to unwind)and subsequently reseals the break before dissociating from the DNAstrand. Topisomerase II has a similar mechanism of action which involvesthe induction of DNA strand breaks or the formation of free radicals.

The term “camptothecin compounds” is used to indicate compounds that arerelated to or derived from the parent camptothecin compound which is awater-insoluble alkaloid derived from the Chinese tree Camptothecinacuminata and the Indian tree Nothapodytes foetida.

The term “podophyllotoxin compounds” is used to indicate compounds thatare related to or derived from the parent podophyllotoxin, which isextracted from the mandrake plant.

The term “anti-tumor vinca alkaloids” is used to indicate compounds thatare related to or derived from extracts of the periwinkle plant (Vincarosea).

The term “alkylating agents” encompass a diverse group of chemicals thathave the common feature that they have the capacity to contribute, underphysiological conditions, alkyl groups to biologically vitalmacromolecules such as DNA. With most of the more important agents suchas the nitrogen mustards and the nitrosoureas, the active alkylatingmoieties are generated in vivo after complex degradative reactions, someof which are enzymatic. The most important pharmacological actions ofthe alkylating agents are those that disturb the fundamental mechanismsconcerned with cell proliferation in particular DNA synthesis and celldivision. The capacity of alkylating agents to interfere with DNAfunction and integrity in rapidly proliferating tissues provides thebasis for their therapeutic applications and for many of their toxicproperties.

The term “anti-tumour anthracycline derivatives” comprise antibioticsobtained from the fungus Strep. peuticus var. caesius and theirderivatives, characterised by having a tetracycline ring structure withan unusual sugar, daunosamine, attached by a glycosidic linkage.

Amplification of the human epidermal growth factor receptor 2 protein(HER 2) in primary breast carcinomas has been shown to correlate with apoor clinical prognosis for certain patients. Trastuzumab is a highlypurified recombinant DNA-derived humanized monoclonal IgG1 kappaantibody that binds with high affiniity and specificity to theextracellular domain of the HER2 receptor.

Many breast cancers have estrogen receptors and growth of these tumorscan be stimulated by estrogen. The terms “estrogen receptor antagonists”and “selective estrogen receptor modulators” are used to indicatecompetitive inhibitors of estradiol binding to the estrogen receptor(ER). Selective estrogen receptor modulators, when bound to the ER,induces a change in the three-dimensional shape of the receptor,inhibiting its binding to the estrogen responsive element (ERE) on DNA.

In postmenopausal women, the principal source of circulating estrogen isfrom conversion of adrenal and ovarian androgens (androstenedione andtestosterone) to estrogens (estrone and estradiol) by the aromataseenzyme in peripheral tissues. Estrogen deprivation through aromataseinhibition or inactivation is an effective and selective treatment forsome postmenopausal patients with hormone-dependent breast cancer.

The term “antiestrogen agent” is used herein to include not onlyestrogen receptor antagonists and selective estrogen receptor modulatorsbut also aromatase inhibitors as discussed above.

The term “differentiating agents” encompass compounds that can, invarious ways, inhibit cell proliferation and induce differentiation.Vitamin D and retinoids are known to play a major role in regulatinggrowth and differentiation of a wide variety of normal and malignantcell types. Retinoic acid metabolism blocking agents (RAMBA's) increasethe levels of endogenous retinoic acids by inhibiting the cytochromeP450-mediated catabolism of retinoic acids.

DNA methylation changes are among the most common abnormalities in humanneoplasia. Hypermethylation within the promotors of selected genes isusually associated with inactivation of the involved genes. The term“DNA methyl transferase inhibitors” is used to indicate compounds thatact through pharmacological inhibition of DNA methyl transferase andreactivation of tumour suppressor gene expression.

The term “kinase inhibitors” comprises potent inhibitors of kinases thatare involved in cell cycle progression and programmed cell death(apoptosis).

The term “farnesyltransferase inhibitors” is used to indicate compoundsthat were designed to prevent farnesylation of Ras and otherintracellular proteins. They have been shown to have effect on malignantcell proliferation and survival.

The term “other HDAC inhibitors” comprises but is not limited to:

-   -   short-chain fatty acids for example butyrate, 4-phenylbutyrate        or valproic acid;    -   hydroxamic acids for example suberoylanilide hydroxamic acid        (SAHA), biaryl hydroxamate A-161906, bicyclic        aryl-N-hydroxycarboxamides, pyroxamide, CG-1521, PXD-101,        sulfonamide hydroxamic acid, LAQ-824, trichostatin A (TSA),        oxamflatin, scriptaid, m-carboxy cinnamic acid bishydroxamic        acid, or trapoxin-hydroxamic acid analogue;    -   cyclic tetrapeptides for example trapoxin, apidicin or        depsipeptide;    -   benzamides for example MS-275 or CI-994, or    -   depudecin.

For the treatment of cancer the compounds according to the presentinvention may be administered to a patient as described above, inconjunction with irradiation. Irradiation means ionising radiation andin particular gamma radiation, especially that emitted by linearaccelerators or by radionuclides that are in common use today. Theirradiation of the tumour by radionuclides can be external or internal.

The present invention also relates to a combination according to theinvention of an anti-cancer agent and a HDAC inhibitor according to theinvention.

The present invention also relates to a combination according to theinvention for use in medical therapy for example for inhibiting thegrowth of tumour cells.

The present invention also relates to a combinations according to theinvention for inhibiting the growth of tumour cells.

The present invention also relates to a method of inhibiting the growthof tumour cells in a human subject which comprises administering to thesubject an effective amount of a combination according to the invention.

This invention further provides a method for inhibiting the abnormalgrowth of cells, including transformed cells, by administering aneffective amount of a combination according to the invention.

The other medicinal agent and HDAC inhibitor may be administeredsimultaneously (e.g. in separate or unitary compositions) orsequentially in either order. In the latter case, the two compounds willbe administered within a period and in an amount and manner that issufficient to ensure that an advantageous or synergistic effect isachieved. It will be appreciated that the preferred method and order ofadministration and the respective dosage amounts and regimes for eachcomponent of the combination will depend on the particular othermedicinal agent and HDAC inhibitor being administered, their route ofadministration, the particular tumour being treated and the particularhost being treated. The optimum method and order of administration andthe dosage amounts and regime can be readily determined by those skilledin the art using conventional methods and in view of the information setout herein.

The platinum coordination compound is advantageously administered in adosage of 1 to 500 mg per square meter (mg/m²) of body surface area, forexample 50 to 400 mg/m², particularly for cisplatin in a dosage of about75 mg/m² and for carboplatin in about 300 mg/m² per course of treatment.

The taxane compound is advantageously administered in a dosage of 50 to400 mg per square meter (mg/m²) of body surface area, for example 75 to250 mg/m², particularly for paclitaxel in a dosage of about 175 to 250mg/m² and for docetaxel in about 75 to 150 mg/m² per course oftreatment.

The camptothecin compound is advantageously administered in a dosage of0.1 to 400 mg per square meter (mg/m²) of body surface area, for example1 to 300 mg/m², particularly for irinotecan in a dosage of about 100 to350 mg/m² and for topotecan in about 1 to 2 mg/m² per course oftreatment.

The anti-tumor podophyllotoxin derivative is advantageously administeredin a dosage of 30 to 300 mg per square meter (mg/m²) of body surfacearea, for example 50 to 250 mg/m², particularly for etoposide in adosage of about 35 to 100 mg/m² and for teniposide in about 50 to 250mg/m² per course of treatment.

The anti-tumor vinca alkaloid is advantageously administered in a dosageof 2 to 30 mg per square meter (mg/m²) of body surface area,particularly for vinblastine in a dosage of about 3 to 12 mg/m², forvincristine in a dosage of about 1 to 2 mg/m², and for vinorelbine indosage of about 10 to 30 mg/m² per course of treatment.

The anti-tumor nucleoside derivative is advantageously administered in adosage of 200 to 2500 mg per square meter (mg/m²) of body surface area,for example 700 to 1500 mg/m², particularly for 5-FU in a dosage of 200to 500 mg/m², for gemcitabine in a dosage of about 800 to 1200 mg/m² andfor capecitabine in about 1000 to 2500 mg/m² per course of treatment.

The alkylating agents such as nitrogen mustard or nitrosourea isadvantageously administered in a dosage of 100 to 500 mg per squaremeter (mg/m²) of body surface area, for example 120 to 200 mg/m²,particularly for cyclophosphamide in a dosage of about 100 to 500 mg/m²,for chlorambucil in a dosage of about 0.1 to 0.2 mg/kg, for carmustinein a dosage of about 150 to 200 mg/m², and for lomustine in a dosage ofabout 100 to 150 mg/m² per course of treatment.

The anti-tumor anthracycline derivative is advantageously administeredin a dosage of 10 to 75 mg per square meter (mg/m²) of body surfacearea, for example 15 to 60 mg/m², particularly for doxorubicin in adosage of about 40 to 75 mg/m², for daunorubicin in a dosage of about 25to 45 mg/m², and for idarubicin in a dosage of about 10 to 15 mg/m² percourse of treatment.

Trastuzumab is advantageously administered in a dosage of 1 to 5 mg persquare meter (mg/m²) of body surface area, particularly 2 to 4 mg/m² percourse of treatment.

The antiestrogen agent is advantageously administered in a dosage ofabout 1 to 100 mg daily depending on the particular agent and thecondition being treated. Tamoxifen is advantageously administered orallyin a dosage of 5 to 50 mg, preferably 10 to 20 mg twice a day,continuing the therapy for sufficient time to achieve and maintain atherapeutic effect. Toremifene is advantageously administered orally ina dosage of about 60 mg once a day, continuing the therapy forsufficient time to achieve and maintain a therapeutic effect.Anastrozole is advantageously administered orally in a dosage of about 1mg once a day. Droloxifene is advantageously administered orally in adosage of about 20-100 mg once a day. Raloxifene is advantageouslyadministered orally in a dosage of about 60 mg once a day. Exemestane isadvantageously administered orally in a dosage of about 25 mg once aday.

These dosages may be administered for example once, twice or more percourse of treatment, which may be repeated for example every 7, 14, 21or 28 days.

In view of their useful pharmacological properties, the components ofthe combinations according to the invention, i.e. the other medicinalagent and the HDAC inhibitor may be formulated into variouspharmaceutical forms for administration purposes. The components may beformulated separately in individual pharmaceutical compositions or in aunitary pharmaceutical composition containing both components.

The present invention therefore also relates to a pharmaceuticalcomposition comprising the other medicinal agent and the HDAC inhibitortogether with one or more pharmaceutical carriers.

The present invention also relates to a combination according to theinvention in the form of a pharmaceutical composition comprising ananti-cancer agent and a HDAC inhibitor according to the inventiontogether with one or more pharmaceutical carriers.

The present invention further relates to the use of a combinationaccording to the invention in the manufacture of a pharmaceuticalcomposition for inhibiting the growth of tumour cells.

The present invention further relates to a product containing as firstactive ingredient a HDAC inhibitor according to the invention and assecond active ingredient an anticancer agent, as a combined preparationfor simultaneous, separate or sequential use in the treatment ofpatients suffering from cancer.

EXPERIMENTAL PART

The following examples are provided for purposes of illustration.

Hereinafter “DCM” means dichloromethane, “DMA” means dimethylacetamide,“DMF” means dimethylformamide, “EtOAc” means ethyl acetate, “iPrOH”means isopropyl, “MeOH” means methanol, “EtOAc” means ethyl acetate,“EtOH” means ethanol, “TEA” means triethylamine, “TFA” meanstrifluoroacetic acid and “THF” means tetrahydrofuran.

A. Preparation of the Intermediates Example A1 a) Preparation of

A solution of 2-naphthalenesulfonyl chloride (0.0094 mol) in DCM (10 ml)was added at 5° C. to a mixture of2-(4-piperidinyl)-1H-isoindole-1,3(2H)-dione (0.0078 mol) and TEA(0.0109 mol) in DCM (20 ml) under N₂ flow. The mixture was kept at roomtemperature for a week-end. Potassium carbonate 10% was added. Themixture was extracted with DCM. The organic layer was separated, dried(MgSO₄), filtered, and the solvent was evaporated till dryness, yielding3.3 g (100%) of intermediate 1.

b) Preparation of

A mixture of intermediate 1 (0.0078 mol) in hydrazine monohydrate (3.5ml) and EtOH (35 ml) was stirred and refluxed for 1 hour. Satured sodiumchloride was added. The mixture was extracted with DCM. The organiclayer was separated, dried (MgSO₄), filtered, and the solvent wasevaporated till dryness, yielding 2g (92%) of intermediate 2, meltingpoint 135° C.

c) Preparation of

A mixture of intermediate 2 (0.0036 mol), 6-chloro-3-pyridinecarboxylicacid, ethyl ester (0.0043 mol) and sodium carbonate (0.0054 ml) in DMA(10 ml) was stirred at 130° C. for 18 hours, poured out into water andextracted with DCM. The organic layer was separated, dried (MgSO₄),filtered, and the solvent was evaporated till dryness. The residue (2.5g) was purified by column chromatography over silica gel (15-40 μm)(eluent: DCM/MeOH 99.5/0.5;). The pure fractions were collected and thesolvent was evaporated. The residue (0.68 g, 43%) was crystallized fromdiethyl ether. The precipitate was filtered off and dried, yielding0.477 g (30%) of intermediate 3, melting point 215° C.

Example A2 a) Preparation of

TEA (0.0112 mol) then a solution of [1,1′-biphenyl]-4-sulfonyl chloride(0.0112 mol) in DCM (5 ml) were added at 5° C. to a mixture of1,4-dioxa-8-azaspiro[4.6]undecane (0.01 mol) in DCM (10 ml) under N₂flow. The mixture was stirred at room temperature for 18 hours.Potassium carbonate 10% was added. The mixture was extracted with DCM.The organic layer was separated, dried (MgSO₄), filtered, and thesolvent was evaporated till dryness, yielding 5.1 g (>100%) ofintermediate 4.

b) Preparation of

A mixture of intermediate 4 (0.01 mol) in HCl 3N (40 ml) and MeOH (20ml) was stirred and refluxed, then cooled, poured out on ice, basifiedwith NH₄OH conc. and extracted with DCM. The organic layer wasseparated, dried (MgSO₄), filtered, and the solvent was evaporated tilldryness, yielding 3.4 g (100%) of intermediate 5.

c) Preparation of

Sodium hydroborate (0.011 mol) was added portionwise at 5° C. to amixture of intermediate 5 (0.01 mol) in MeOH (35 ml) under N₂ flow. Themixture was kept at room temperature for 1 hour, poured out into waterand extracted with DCM. The organic layer was separated, dried (MgSO₄),filtered, and the solvent was evaporated till dryness, yielding 3.2 g(97%) of intermediate 6.

d) Preparation of

Diazenedicarboxylic acid, bis(1-methylethyl) ester (0.0126 mol) wasadded dropwise to a mixture of intermediate 6 (0.0096 mol),1H-isoindole-1,3(2H)-dione (0.0126 mol) and triphenyl-phosphine (0.0126mol) in THF (35 ml) under N₂ flow. The mixture was kept at roomtemperature for 18 hours. Potassium carbonate 10% was added. The mixturewas extracted with DCM. The organic layer was separated, dried (MgSO₄),filtered, and the solvent was evaporated till dryness. The residue (13.1g) was purified by column chromatography over silica gel (15-40 μm)(eluent: DCM/EtOAc 99/1). The pure fractions were collected and thesolvent was evaporated, yielding 2.9 g (66%) of intermediate 7.

e) Preparation of

A mixture of intermediate 7 (0.0063 mol) in hydrazine monohydrobromide(2.9 ml) and EtOH (30 ml) was stirred and refluxed for 1 hour, thencooled. Satured NaCl was added. The mixture was extracted with DCM. Theorganic layer was separated, dried (MgSO₄), filtered, and the solventwas evaporated till dryness. The residue (2.5 g) was crystallized fromacetonitrile/diethyl ether. The precipitate was filtered off and dried,yielding 0.89 g (89%) of intermediate 8, melting point 141° C.

Preparation of

Intermediate 10 was handled analogously as described in example A2 (c,d, e) to give 0.157 g (52%) of intermediate 19, melting point 123° C.

Example A3 a) Preparation of

TEA (0.0112 mol) then a solution of 2-naphthalenesulfonyl chloride(0.0112 mol) in DCM (5 ml) were added at 5° C. to a mixture of1,4-dioxa-8-azaspiro[4.6]undecane (0.01 mol) in DCM (10 ml) under N₂flow. The mixture was stirred at room temperature for 18 hours.Potassium carbonate 10% was added. The mixture was extracted with DCM.The organic layer was separated, dried (MgSO₄), filtered, and thesolvent was evaporated till dryness, yielding 3.9 g (>100%) ofintermediate 9.

b) Preparation of

A mixture of intermediate 9 (0.01 mol) in hydrochloric acid (35 ml) andMeOH (35 ml) was stirred and refluxed for 30 minutes, poured out on ice,basified with NH₄OH and extracted with DCM. The organic layer wasseparated, dried (MgSO₄), filtered, and the solvent was evaporated tilldryness, yielding 3.4 g (>100%) of intermediate 10.

c) Preparation of

Tetraisopropanolatotitanium (0.012 mol) was added at room temperature toa mixture of intermediate 10 (0.01 mol) and benzenemethanamine (0.011mol) in EtOH (40 ml). The mixture was stirred for 18 hours. Sodiumhydroborate (0.011 mol) was added portionwise. The mixture was stirredat room temperature for 1 hour and 30 minutes. Potassium carbonate 10%was added. The mixture was extracted with DCM. The salts were filteredand washed with DCM. The organic layer was separated, dried (MgSO₄),filtered, and the solvent was evaporated till dryness, yielding 2.7 g(70%) of intermediate 11.

d) Preparation of

A mixture of intermediate 11 (0.0068 mol) and Pd/C 10% (0.5 g) in aceticacid (3 ml) and MeOH (30 ml) was hydrogenated at 50° C. for 48 hoursunder a 3 bar pressure, then filtered and washed with DCM. The filtratewas evaporated, yielding: 2.55 g (>100%) of intermediate 12.

Example A4 a) Preparation of

Sodium hydride 60% (0.008 mol) was added portionwise at 0° C. to amixture of 4-(aminomethyl)-1-piperidinecarboxylic acid,1,1-dimethylethyl ester (0.004 mol) in THF (20 ml) under N₂ flow. Themixture was stirred at 0° C. for 1 hour. A solution of2-(methylsulfonyl)-5-pyrimidinecarboxylic acid, ethyl ester (0.0052 mol)in THF (10 ml) was added dropwise at 0° C. The mixture was brought toroom temperature, stirred for 2 hours, poured out into ice water andextracted with EtOAc. The organic layer was separated, dried (MgSO₄),filtered, and the solvent was evaporated. The residue (1.5 g) waspurified by column chromatography over silica gel (15-40 μm) (eluent:DCM/MeOH/NH₄OH 98/2/0.1). The pure fractions were collected and thesolvent was evaporated. The residue (0.5 g, 35%) was crystallized fromDIPE. The precipitate was filtered off and dried, yielding 0.28 g ofintermediate 13, melting point 110° C.

b) Preparation of

A mixture of intermediate 13 (0.016 mol) in hydrochloric acid 3N (60 ml)and THF (15 ml) was stirred at 80° C. for 6 hours, poured out into icewater, basified with NH₄OH and extracted with DCM. The organic layer wasseparated, dried (MgSO₄), filtered, and the solvent was evaporated,yielding 1.4 g (33%) of intermediate 14.

Example A5 a) Preparation of

A solution of 1-hydroxy-1H-benzotriazole (0.011 mol) in DCM (5 ml) wasadded at 5° C. to a mixture of (3-piperidinylmethyl)- carbamic acid,1,1-dimethylethyl ester (0.01 mol) and TEA (0.014 mol) in DCM (5 ml)under N₂ flow. The mixture was stirred at room temperature for 18 hours.Water was added. The mixture was extracted with DCM. The organic layerwas separated, dried (MgSO₄), filtered, and the solvent was evaporatedtill dryness, yielding 4g (100%) of intermediate 15.

b) Preparation of

A mixture of intermediate 15 (0.01 mol) in HCl/2-propanol (50 ml) wasstirred at 50° C. for 15 minutes, basified with NH₄OH concentrated andthe solvent was evaporated till dryness. The residue was taken up in DCMand filtered. The filtrate was dried (MgSO₄), filtered and the solventwas evaporated till dryness, yielding 0.53 g (18%) of intermediate 16.

Example A6 a) Preparation of

Sodium hydride 60% (0.014 mol) was added portionwise at 0° C. to amixture of (2-morpholinylmethyl)-carbamic acid, 1,1-dimethylethyl ester(0.0092 mol) in THF (30 ml) under N₂ flow. The mixture was stirred at 0°C. for 1 hour. A solution of 2-naphthalenesulfonyl chloride (0.0111 mol)in THF (30 ml) was added. The mixture was brought to room temperatureovernight, poured out into ice water and extracted with EtOAc. Theorganic layer was separated, dried (MgSO₄), filtered, and the solventwas evaporated. The residue (4.9 g) was purified by columnchromatography over silica gel (15-40 μm) (eluent: cyclohexane/EtOAc70/30). The pure fractions were collected and the solvent wasevaporated, yielding 2.15 g (58%) of intermediate 17.

b) Preparation of

A mixture of intermediate 17 (0.0052 mol) in HCl 6N (25 ml) was stirredat 80° C. for 12 hours, poured out on ice, basified with sodiumhydroxide and extracted with DCM. The organic layer was separated, dried(MgSO₄), filtered, and the solvent was evaporated, yielding 1 g (63%) ofintermediate 18.

B. Preparation of the Final Compounds Example B1 a) Preparation of

A mixture of intermediate 3 (0.0014 mol) and sodium hydroxide (0.0028mol) in EtOH (10 ml) was stirred and refluxed for 2 hours, then cooled.The precipitate was filtered, washed with EtOH, then with diethyl etherand dried, yielding 0.5 g (82%) of intermediate 20.

b) Preparation of

N′-(ethylcarbonimidoyl)-N,N-dimethyl-1,3-propanediamine,monohydrochloride (0.0013 mol) was added to a mixture of intermediate 20(0.0011 mol), 0-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.0013 mol)and 1-hydroxy-1H-benzotriazole (0.0013 mol) in DCM/THF (10 ml) under N₂flow. The mixture was stirred at room temperature overnight. Potassiumcarbonate 10% was added. The mixture was extracted with DCM. The organiclayer was separated, dried (MgSO₄), filtered, and the solvent wasevaporated till dryness. The residue (0.95 g) was purified by columnchromatography over silica gel (15-40 μm)(eluent: DCM/MeOH/NH₄OH97/3/0.2). The pure fractions were collected and the solvent wasevaporated. The residue (0.48 g, 82%) was crystallized fromacetonitile/diethyl ether. The precipitate was filtered off and dried,yielding 0.455 g (77%) of intermediate 21, melting point 129° C.

c) Preparation of

Trifluoroacetic acid (0.5 ml) was added to a mixture of intermediate 21(0.0007 mol) in MeOH (5 ml). The mixture was stirred at room temperaturefor 18 hours. Trifluoroacetic acid (0.5 ml) was added. The mixture wasstirred at room temperature for 18 hours. The solvent was evaporatedtill dryness. The residue was crystallized from MeOH/DCM/diethyl ether.The precipitate was filtered off and dried, yielding 0.195 g (48%) ofcompound 1 .0.79 C₂HF₃O₂, melting point 160° C.

Example B2 Preparation of

A mixture of intermediate 8 (0.0031 mol), 6-chloro-3-pyridinecarboxylicacid, ethyl ester (0.0037 mol) and sodium carbonate (0.0046 mol) in DMA(10 ml) was stirred at 130° C. for 18 hours. Water was added. Themixture was extracted several times with EtOAc. The organic layer wasseparated, dried (MgSO₄), filtered, and the solvent was evaporated tilldryness. The residue (2.1 g) was purified by column chromatography oversilica gel (15-40 μm) (eluent: DCM/MeOH 99/1). The pure fractions werecollected and the solvent was evaporated. The residue (0.414 g, 28%) wascrystallized from diethyl ether. The precipitate was filtered off anddried, yielding 0.294 g (20%) of intermediate 22, melting point 176° C.

Intermediate 22 was handled analogously as described in example [B1] togive 0.084 g (61%) of compound 2 .1.2 H₂O .0.71 C₂HF₃O₂ (1:1), meltingpoint 115° C.

Example B3 Preparation of

Sodium hydride 60% in oil (0.0045 mol) was added at 5° C. to a mixtureof intermediate 19 (0.003 mol) in THF (20 ml) under N₂ flow. The mixturewas kept for 1 hour. A solution of2-(methylsulfonyl)-5-pyrimidinecarboxylic acid, ethyl ester (0.0039 mol)in THF (10 ml) was added. The mixture was stirred at 5° C. for 2 hours.Water was added. The mixture was extracted twice with DCM. The organiclayer was separated, dried (MgSO₄), filtered, and the solvent wasevaporated till dryness. The residue (1.5 g) was purified by columnchromatography over silica gel (15-40 μm) (eluent: DCM/MeOH 99/1). Thepure fractions were collected and the solvent was evaporated. Theresidue (0.47 g, 34%) was crystallized from diethyl ether. Theprecipitate was filtered off and dried, yielding 0.224 g (16%) ofintermediate 23, melting point 186° C.

Intermediate 23 was handled analogously as described in example [B1] togive 0.032 g (26%) of compound 3 .0.22 C₂HF₃O₂, melting point 140° C.

Example B4 Preparation of

Sodium hydride (0.0038 mol) was added at 5° C. to a mixture ofintermediate 8 (0.0025 mol) in THF (20 ml) under N₂ flow. The mixturewas kept for 1 hour. A solution of2-(methylsulfonyl)-5-pyrimidinecarboxylic acid, ethyl ester (0.0033 mol)in THF (10 ml) was added. The mixture was stirred at 5° C. for 18 hours.Water was added. The mixture was extracted with DCM. The organic layerwas separated, dried (MgSO₄), filtered, and the solvent was evaporatedtill dryness. The residue (1.1 g) was purified by column chromatographyover silica gel (15-35 μm) (eluent: cyclohexane/EtOAc 70/30). The purefractions were collected and the solvent was evaporated. The residue(0.18 g, 15%) was crystallized from diethyl ether. The precipitate wasfiltered off and dried, yielding 0.141 g (12%) of intermediate 24,melting point 151° C.

Intermediate 24 was handled analogously as described in example [B1] togive 0.04 g (57%) of compound 4.C₂HF₃O₂, melting point 227° C.

Example B5 Preparation of

Sodium hydride 60% in oil (0.0045 mol) was added at 5° C. to a mixtureof intermediate 2 (0.003 mol) in THF (15 ml) under N₂ flow. The mixturewas kept for 1 hour. A solution of2-(methylsulfonyl)-5-pyrimidinecarboxylic acid, ethyl ester (0.0039 mol)in THF (10 ml) was added. The mixture was stirred at 5° C. for 2 hours.Water was added. The mixture was extracted twice with DCM. The organiclayer was separated, dried (MgSO₄), filtered, and the solvent wasevaporated till dryness. The residue (1.4 g) was purified by columnchromatography over silica gel (15-40 μm) (eluent: DCM/MeOH 99/1). Thepure fractions were collected and the solvent was evaporated. Theresidue (0.49 g, 37%) was crystallized from acetonitrile. Theprecipitate was filtered, washed with diethyl ether and dried, yielding0.172 g (13%) of intermediate 25, melting point 226° C.

Intermediate 25 was handled analogously as described in example [B1] togive 0.064 g (49%) of compound 5 .0.17 C₂HF₃O₂, melting point 256° C.

Example B6 Preparation of

A mixture of intermediate 12 (0.0052 mol), 6-chloro-3-pyridinecarboxylicacid, ethyl ester (0.0062 mol) and sodium carbonate (0.0078 mol) in DMA(20 ml) was stirred at 130° C. for 18 hours. Water was added. Themixture was extracted with EtOAc. The organic layer was separated, dried(MgSO₄), filtered, and the solvent was evaporated till dryness. Theresidue (2.6 g) was purified by column chromatography over silica gel915-40 μm) (eluent: DCM/MeOH 99/1;). The pure fractions were collectedand the solvent was evaporated, yielding 0.64 g (27%) of intermediate26, melting point 146° C. Intermediate 26 was handled analogously asdescribed in example [B1] to give 0.263 g (67%) of compound 6 H₂O(1:1).0.73 C₂HF₃O₂, melting point 115° C.

Example B7 b) Preparation of

A solution of 2-naphthalenesulfonyl chloride (0.0006 mol) in DCM (2 ml)was added dropwise at 0° C. to a mixture of intermediate 14 (0.0005 mol)and TEA (0.0008 mol) in DCM (3 ml). The mixture was stirred at roomtemperature for 12 hours, poured out into water and extracted withEtOAc. The organic layer was separated, dried (MgSO₄), filtered, and thesolvent was evaporated. The residue (0.33 g) was purified by columnchromatography over silica gel (10 μm) (eluent: DCM 100). The purefractions were collected and the solvent was evaporated, yielding 0.21 g(80%) of intermediate 27. Intermediate 27 was handled analogously asdescribed in example [B1] to give 0.043 g (33%) of compound 7, meltingpoint 240° C.

Example B8 Preparation of

Sodium hydride 60% in oil (0.0023 mol) was added portionwise at 5° C. toa mixture of intermediate 16 (0.0017 mol) in THF (5 ml) under N₂ flow.The mixture was stirred for 30 minutes. A solution of2-(methylsulfonyl)-5-pyrimidinecarboxylic acid, ethyl ester (0.0021 mol)in THF (2 ml) was added. The mixture was stirred at room temperature for18 hours. Potassium carbonate 10% was added. The mixture was extractedwith DCM. The organic layer was separated, dried (MgSO₄), filtered, andthe solvent was evaporated till dryness. The residue (0.72 g) waspurified by column chromatography over silica gel (15-40 μm) (eluent:DCM/MeOH 99/1). The pure fractions were collected and the solvent wasevaporated, yielding 0.3 g (39%) of intermediate 28. Intermediate 28washandled analogously as described in example [B1] to give 0.119 g (75%)of compound 8, melting point 137° C.

Example B9 Preparation of

A mixture of intermediate 18 (0.0019 mol),2-(methylsulfonyl)-5-pyrimidinecarboxylic acid, ethyl ester (0.0025 mol)and potassium carbonate (0.0039 mol) in acetonitrile (15 ml) was stirredat room temperature for 12 hours, poured out into ice water andextracted with EtOAc. The organic layer was washed with water, dried(MgSO₄), filtered, and the solvent was evaporated. The residue (1g) waspurified by column chromatography over silica gel (15-40 μm) (eluent:DCM/EtOAc 80/20). The pure fractions were collected and the solvent wasevaporated. The residue (0.48 g, 89%) was crystallized fromCH₃CN/diethyl ether. The precipitate was filtered off and dried,yielding 0.2 g of intermediate 29, melting point 168° C.

Intermediate 29 was handled analogously as described in example [B1] togive 0.08 g (73%) of compound 9, melting point 226° C.

Table F-1 lists the compounds that were prepared according to one of theabove Examples. The following abbreviations were used in the tables:.C₂HF₃O₂ stands for the trifluoroacetate salt.

TABLE F-1

•0.79 C₂HF₃O₂; Co. No. 1; Ex. [B1]; mp. 160° C.

•0.83 C₂HF₃O₂; Co. No. 10; Ex. [B1]; mp. 182° C.

•0.84 C₂HF₃O₂; Co. No. 11; Ex. [B1]; mp. 188° C.

•1.2 H₂O •0.71 C₂HF₃O₂; Co. No. 2; Ex. [B2]; mp. 115° C.

•2 H₂O •0.53 C₂HF₃O₂; Co. No. 12; Ex. [B2]; mp. 147° C.

Co. No. 13; Ex. [B2]; mp. 213° C.

•0.22 C₂HF₃O₂; Co. No. 3; Ex. [B3]; mp. 140° C.

Co. No. 14; Ex. [B3]; mp. 250° C.

•C₂HF₃O₂; Co. No. 4; Ex. [B4]; mp. 227° C.

•0.17 C₂HF₃O₂; Co. No. 5; Ex. [B5]; mp. 256° C.

•H₂O •0.73 C₂HF₃O₂; Co. No. 6; Ex. [B6]; mp. 146° C.

•2.27 C₂HF₃O₂; Co. No. 15; Ex. [B6]; mp. 192° C.

Co. No. 7; Ex. [B7]; mp. 240° C.

Co. No. 8; Ex. [B8]; mp. 137° C.

Co. No. 9; Ex. [B9]; mp. 226° C.

C. Pharmacological Example:

The in vitro assay for inhibition of histone deacetylase (see exampleC.1) measures the inhibition of HDAC enzymatic activity obtained withthe compounds of formula (I).

Cellular activity of the compounds of formula (I) was determined onA2780 tumour cells using a colorimetric assay for cell toxicity orsurvival (Mosmann Tim, Journal of Immunological Methods 65: 55-63,1983)(see example C.2).

Kinetic solubility in aqueous media measures the ability of a compoundto stay in aqueous solution upon dilution (see example C.3).

DMSO-stock solutions are diluted with a single aqueous buffer solvent in3 consecutive steps. For every dilution turbidity is measured with anephelometer.

Metabolism of drugs means that a lipid-soluble xenobiotic or endobioticcompound is enzymatically transformed into (a) polar, water-soluble, andexcretable metabolite(s). The major organ for drug metabolism is theliver. The metabolic products are often less active than the parent drugor inactive. However, some metabolites may have enhanced activity ortoxic effects. Thus drug metabolism may include both “detoxication” and“toxication” processes. One of the major enzyme systems that determinethe organism's capability of dealing with drugs and chemicals isrepresented by the cytochrome P450 monooxygenases, which are NADPHdependent enzymes. Metabolic stability of compounds can be determined invitro with the use of subcellular human tissue (see example C.4). Heremetabolic stability of the compounds is expressed as % of drugmetabolised after 15 minutes incubation of these compounds withmicrosomes. Quantitation of the compounds was determined by LC-MSanalysis.

The tumour suppressor p53 transcriptionally activates a number of genesincluding the WAF1/CIP1 gene in response to DNA damage. The 21 kDaproduct of the WAF1 gene is found in a complex involving cyclins, cyclindependent kinases (CDKs), and proliferating cell nuclear antigen (PCNA)in normal cells but not transformed cells and appears to be a universalinhibitor of CDK activity. One consequence of p21WAF1 binding to andinhibiting CDKs is to prevent CDK-dependent phosphorylation andsubsequent inactivation of the Rb protein, which is essential for cellcycle progression. Induction of p21WAF1 in response to cellular contactwith a HDAC inhibitor is therefore a potent and specific indicator ofinhibition of cell cycle progression at both the G1 and G2 checkpoints.

The capacity of the compounds to induce p21WAF1 was measured with thep21WAF1 enzyme linked immunosorbent assay (WAF1 ELISA of Oncogene). Thep21 WAF1 assay is a “sandwich” enzyme immunoassay employing both mousemonoclonal and rabbit polyclonal antibodies. A rabbit polyclonalantibody, specific for the human WAF1 protein, has been immobilized ontothe surface of the plastic wells provided in the kit. Any p21WAF presentin the sample to be assayed will bind to the capture antibody. Thebiotinylated detector monoclonal antibody also recognizes human p21WAF1protein, and will bind to any p21WAF1, which has been retained by thecapture antibody. The detector antibody, in turn, is bond by horseradishperoxidas-conjugated streptavidin. The horseradish peroxidase catalysesthe conversion of the chromogenic substrate tetra-methylbenzidine from acolorless solution to a blue solution (or yellow after the addition ofstopping reagent), the intensity of which is proportional to the amountof p21WAF1 protein bond to the plate. The colored reaction product isquantified using a spectrophotometer. Quantitation is achieved by theconstruction of a standard curve using known concentrations of p21 WAF 1(provided lyophilised)(see example C.5).

Example C.1 In Vitro Assay for Inhibition of Histone Deacetylase

HeLa nuclear extracts (supplier: Biomol) were incubated at 60 μg/ml with2×10⁻⁸ M of radiolabeled peptide substrate. As a substrate for measuringHDAC activity a synthetic peptide, i.e. the amino acids 14-21 of histoneH4, was used. The substrate is biotinylated at the NH₂-terminal partwith a 6-aminohexanoic acid spacer, and is protected at theCOOH-terminal part by an amide group and specifically [³H]acetylated atlysine 16. The substrate,biotin-(6-aminohexanoic)Gly-Ala-([³1-1]-acetyl-Lys-Arg-His-Arg-Lys-Val-NH₂),was added in a buffer containing 25 mM Hepes, 1 M sucrose, 0.1 mg/ml BSAand 0.01% Triton X-100 at pH 7.4. After 30 min the deacetylationreaction was terminated by the addition of HCl and acetic acid. (finalconcentration 0.035 mM and 3.8 mM respectively). After stopping thereaction, the free ³H-acetate was extracted with ethylacetate. Aftermixing and centrifugation, the radioactivity in an aliquot of the upper(organic) phase was counted in a β-counter.

For each experiment, controls (containing HeLa nuclear extract and DMSOwithout compound), a blank incubation (containing DMSO but no HeLanuclear extract or compound) and samples (containing compound dissolvedin DMSO and HeLa nuclear extract) were run in parallel. In firstinstance, compounds were tested at a concentration of 10⁻⁵M. When thecompounds showed activity at 10⁻⁵M, a concentration-response curve wasmade wherein the compounds were tested at concentrations between 10⁻⁵Mand 10⁻¹²M. In each test the blank value was subtracted from both thecontrol and the sample values. The control sample represented 100% ofsubstrate deactylation. For each sample the radioactivity was expressedas a percentage of the mean value of the controls. When appropriateIC₅₀-values (concentration of the drug, needed to reduce the amount ofmetabolites to 50% of the control) were computed using probit analysisfor graded data. Herein the effects of test compounds are expressed aspIC₅₀ (the negative log value of the IC₅₀-value). All tested compoundsshowed enzymatic activity at a test concentration of 10⁻⁵M and 14compounds had a pIC₅₀ 5 (see table F-2).

Example C.2 Determination of Antiproliferative Activity on A2780 Cells

All compounds tested were dissolved in DMSO and further dilutions weremade in culture medium. Final DMSO concentrations never exceeded 0.1%(v/v) in cell proliferation assays. Controls contained A2780 cells andDMSO without compound and blanks contained DMSO but no cells. MTT wasdissolved at 5 mg/ml in PBS. A glycine buffer comprised of 0.1 M glycineand 0.1 M NaCl buffered to pH 10.5 with NaOH (1 N) was prepared (allreagents were from Merck).

The human A2780 ovarian carcinoma cells (a kind gift from Dr. T. C.Hamilton [Fox Chase Cancer Centre, Pennsylvania, USA]) were cultured inRPMI 1640 medium supplemented with 2 mM L-glutamine, 50 μg/ml gentamicinand 10% fetal calf serum. Cells were routinely kept as monolayercultures at 37° C. in a humidified 5% CO₂ atmosphere. Cells werepassaged once a week using a trypsin/EDTA solution at a split ratio of1:40. All media and supplements were obtained from Life Technologies.Cells were free of mycoplasma contamination as determined using theGen-Probe Mycoplasma Tissue Culture kit (supplier: BioMérieux).

Cells were seeded in NUNC™ 96-well culture plates (Supplier: LifeTechnologies) and allowed to adhere to the plastic overnight. Densitiesused for plating were 1500 cells per well in a total volume of 200 μlmedium. After cell adhesion to the plates, medium was changed and drugsand/or solvents were added to a final volume of 200 μl. Following fourdays of incubation, medium was replaced by 200 μl fresh medium and celldensity and viability was assessed using an MTT-based assay. To eachwell, 25 μl MTT solution was added and the cells were further incubatedfor 2 hours at 37° C. The medium was then carefully aspirated and theblue MTT-formazan product was solubilized by addition of 25 μl glycinebuffer followed by 100 μl of DMSO. The microtest plates were shaken for10 min on a microplate shaker and the absorbance at 540 nm was measuredusing an Emax 96-well spectrophotometer (Supplier: Sopachem). Within anexperiment, the results for each experimental condition are the mean of3 replicate wells. For initial screening purposes, compounds were testedat a single fixed concentration of 10⁻⁶ M. For active compounds, theexperiments were repeated to establish full concentration-responsecurves. For each experiment, controls (containing no drug) and a blankincubation (containing no cells or drugs) were run in parallel. Theblank value was subtracted from all control and sample values. For eachsample, the mean value for cell growth (in absorbance units) wasexpressed as a percentage of the mean value for cell growth of thecontrol. When appropriate, IC₅₀-values (concentration of the drug,needed to reduce cell growth to 50% of the control) were computed usingprobit analysis for graded data (Finney, D. J., Probit Analyses, 2^(nd)Ed. Chapter 10, Graded Responses, Cambridge University Press, Cambridge1962). Herein the effects of test compounds are expressed as pIC₅₀ (thenegative log value of the IC₅₀-value). Most of the tested compoundsshowed cellular activity at a test concentration of 10⁻⁶ M and 14compounds had a pIC₅₀≧5 (see table F-2).

Example C.3 Kinetic Solubility in Aqueous Media

In the first dilution step, 10 μl of a concentrated stock-solution ofthe active compound, solubilized in DMSO (5 mM), was added to 100 μlphosphate citrate buffer pH 7.4 and mixed. In the second dilution step,an aliquot (20 μl) of the first dilution step was further dispensed in100 μl phosphate citrate buffer pH 7.4 and mixed. Finally, in the thirddilution step, a sample (20 μl) of the second dilution step was furtherdiluted in 100 μl phosphate citrate buffer pH 7.4 and mixed. Alldilutions were performed in 96-well plates Immediately after the lastdilution step the turbidity of the three consecutive dilution steps weremeasured with a nephelometer. Dilution was done in triplicate for eachcompound to exclude occasional errors. Based on the turbiditymeasurements a ranking is performed into 3 classes. Compounds with highsolubility obtained a score of 3 and for this compounds the firstdilution is clear. Compounds with medium solubility obtained a score of2. For these compounds the first dilution is unclear and the seconddilution is clear. Compounds with low solubility obtained a score of 1and for these compounds both the first and the second dilution areunclear. The solubility of 14 compounds was measured. From thesecompounds 6 showed a score of 3, 2 had a score of 2 and 6 demonstrated ascore of 1 (see table F-2).

Example C.4 Metabolic Stability

Sub-cellular tissue preparations were made according to Gorrod et al.(Xenobiotica 5: 453-462, 1975) by centrifugal separation aftermechanical homogenization of tissue. Liver tissue was rinsed in ice-cold0.1 M Tris-HCl (pH 7.4) buffer to wash excess blood. Tissue was thenblotted dry, weighed and chopped coarsely using surgical scissors. Thetissue pieces were homogenized in 3 volumes of ice-cold 0.1 M phosphatebuffer (pH 7.4) using either a Potter-S (Braun, Italy) equipped with aTeflon pestle or a Sorvall Omni-Mix homogeniser, for 7×10 sec. In bothcases, the vessel was kept in/on ice during the homogenization process.

Tissue homogenates were centrifuged at 9000×g for 20 minutes at 4° C.using a Sorvall centrifuge or Beckman Ultracentrifuge. The resultingsupernatant was stored at −80° C. and is designated ‘S9’.

The S9 fraction can be further centrifuged at 100.000×g for 60 minutes(4° C.) using a Beckman ultracentrifuge. The resulting supernatant wascarefully aspirated, aliquoted and designated ‘cytosol’. The pellet wasre-suspended in 0.1 M phosphate buffer (pH 7.4) in a final volume of 1ml per 0.5 g original tissue weight and designated ‘microsomes’.

All sub-cellular fractions were aliquoted, immediately frozen in liquidnitrogen and stored at −80° C. until use.

For the samples to be tested, the incubation mixture contained PBS(0.1M), compound (5 μM), microsomes (1 mg/ml) and a NADPH-generatingsystem (0.8 mM glucose-6-phosphate, 0.8 mM magnesium chloride and 0.8Units of glucose-6-phosphate dehydrogenase). Control samples containedthe same material but the microsomes were replaced by heat inactivated(10 mM at 95 degrees Celsius) microsomes. Recovery of the compounds inthe control samples was always 100%.

The mixtures were preincubated for 5 mM at 37 degrees Celsius. Thereaction was started at timepoint zero (t=0) by addition of 0.8 mM NADPand the samples were incubated for 15 min (t=15). The reaction wasterminated by the addition of 2 volumes of DMSO. Then the samples werecentrifuged for 10 min at 900×g and the supernatants were stored at roomtemperature for no longer as 24 h before analysis. All incubations wereperformed in duplo. Analysis of the supernatants was performed withLC-MS analysis. Elution of the samples was performed on a Xterra MS C18(50×4.6 mm, 5 μm, Waters, US). An Alliance 2790 (Supplier: Waters, US)HPLC system was used. Elution was with buffer A (25 mM ammoniumacetate(pH 5.2) in H₂O/acetonitrile (95/5)), solvent B being acetonitrile andsolvent C methanol at a flow rate of 2.4 ml/min. The gradient employedwas increasing the organic phase concentration from 0% over 50% B and50% C in 5 min up to 100% B in 1 min in a linear fashion and organicphase concentration was kept stationary for an additional 1.5 min. Totalinjection volume of the samples was 25 μl.

A Quattro (supplier: Micromass, Manchester, UK) triple quadrupole massspectrometer fitted with and ESI source was used as detector. The sourceand the desolvation temperature were set at 120 and 350° C. respectivelyand nitrogen was used as nebuliser and drying gas. Data were acquired inpositive scan mode (single ion reaction). Cone voltage was set at 10 Vand the dwell time was 1 sec. Metabolic stability was expressed as %metabolism of the compound after 15 min of incubation in the presence ofactive microsomes

$\left( {E({act})} \right)\left( {{\% \mspace{14mu} {metabolism}} = {{100\%} - {\left( {\left( \frac{{{Total}\mspace{14mu} {Ion}\mspace{14mu} {Current}\mspace{14mu} ({TIC})\mspace{14mu} {of}\mspace{14mu} {E({act})}\mspace{14mu} {at}\mspace{14mu} t} = 15}{{{TIC}\mspace{14mu} {of}\mspace{14mu} {E({act})}\mspace{14mu} {at}\mspace{14mu} t} = 0} \right) \times 100} \right).}}} \right.$

Compounds that had a percentage metabolism less than 20% were defined ashighly metabolic stable. Compound that had a metabolism between 20 and70% were defined as intermediately stable and compounds that showed apercentage metabolism higher than 70 were defined as low metabolicstable. Three reference compounds were always included whenever ametabolic stability screening was performed. Verapamil was included as acompound with low metabolic stability (% metabolism=73%). Cisapride wasincluded as a compound with medium metabolic stability (% metabolism45%) and propanol was included as a compound with intermediate to highmetabolic stability (25% metabolism). These reference compounds wereused to validate the metabolic stability assay.

Three compounds were tested. One compound had a percentage metabolismless than 20% and two compounds had a percentage metabolism between 20and 70%.

Example C.5 p21 Induction Capacity

The following protocol has been applied to determine the p21 proteinexpression level in human A2780 ovarian carcinoma cells. The A2780 cells(20000 cells/180 μl) were seeded in 96 microwell plates in RPMI 1640medium supplemented with 2 mM L-glutamine, 50 μg/ml gentamicin and 10%fetal calf serum. 24 hours before the lysis of the cells, compounds wereadded at final concentrations of 10⁻⁵, 10⁻⁶, 10⁻⁷ and 10⁻⁸ M. Allcompounds tested were dissolved in DMSO and further dilutions were madein culture medium. 24 hours after the addition of the compound, thesupernatants were removed from the cells. Cells were washed with 200 μlice-cold PBS. The wells were aspirated and 30 μl of lysisbuffer (50 mMTris.HCl (pH 7.6), 150 mM NaCl, 1% Nonidet p40 and 10% glycerol) wasadded. The plates were incubated overnight at −70° C.

The appropriate number of microtiter wells were removed from the foilpouch and placed into an empty well holder. A working solution (1×) ofthe Wash Buffer (20× plate wash concentrate: 100 ml 20-fold concentratedsolution of PBS and surfactant. Contains 2% chloroacetamide) wasprepared. The lyophilised p21WAF standard was reconstituted withdistilled H₂O and further diluted with sample diluent (provided in thekit).

The samples were prepared by diluting them 1:4 in sample diluent. Thesamples (100 IA) and the p21WAF1 standards (100 μl) were pipetted intothe appropriate wells and incubated at room temperature for 2 hours. Thewells were washed 3 times with 1× wash buffer and then 100 μl ofdetector antibody reagent (a solution of biotinylated monoclonal p21WAF1antibody) was pipetted into each well. The wells were incubated at roomtemperature for 1 hour and then washed three times with 1× wash buffer.The 400× conjugate (peroxidase streptavidine conjugate: 400-foldconcentrated solution) was diluted and 100 μl of the 1× solution wasadded to the wells. The wells were incubated at room temperature for 30min and then washed 3 times with 1× wash buffer and 1 time withdistilled H₂O. Substrate solution (chromogenic substrate)(100 μl) wasadded to the wells and the wells were incubated for 30 minutes in thedark at room temperature. Stop solution was added to each well in thesame order as the previously added substrate solution. The absorbance ineach well was measured using a spectrophotometric plate reader at dualwavelengths of 450/595 nm.

For each experiment, controls (containing no drug) and a blankincubation (containing no cells or drugs) were run in parallel. Theblank value was subtracted from all control and sample values. For eachsample, the value for p21WAF1 induction (in absorbance units) wasexpressed as the percentage of the value for p21WAF1 present in thecontrol. Percentage induction higher than 130% was defined assignificant induction. Three compounds were tested in this assay andshowed significant induction.

TABLE F-2 Table F-2 lists the results of the compounds that were testedaccording to example C.1, C.2, and C.3. Enzyme Cellular activityactivity Solubility Co. No. pIC50 pIC50 Score 15 <5 <5 3 10 6.81 5.396 31 7.676 5.61 3 3 7.306 5.506 2 11 6.594 5.6 1 5 7.525 5.473 3 4 7.3145.838 1 14 7.077 5.201 1 6 6.853 5.345 2 12 7.592 5.718 3 2 6.871 5.8041 13 6.934 5.452 7 7.408 5.109 1 8 7.23 5.721 1 9 7.161 5.667 3

D. Composition Example Film-Coated Tablets Preparation of Tablet Core

A mixture of 100 g of a compound of formula (I), 570 g lactose and 200 gstarch is mixed well and thereafter humidified with a solution of 5 gsodium dodecyl sulphate and 10 g polyvinyl-pyrrolidone in about 200 mlof water. The wet powder mixture is sieved, dried and sieved again. Thenthere is added 100 g microcrystalline cellulose and 15 g hydrogenatedvegetable oil. The whole is mixed well and compressed into tablets,giving 10.000 tablets, each comprising 10 mg of a compound of formula(I).

Coating

To a solution of 10 g methyl cellulose in 75 ml of denaturated ethanolthere is added a solution of 5 g of ethyl cellulose in 150 ml ofdichloromethane. Then there are added 75 ml of dichloromethane and 2.5ml 1,2,3-propanetriol 10 g of polyethylene glycol is molten anddissolved in 75 ml of dichloromethane. The latter solution is added tothe former and then there are added 2.5 g of magnesium octadecanoate, 5g of polyvinyl-pyrrolidone and 30 ml of concentrated colour suspensionand the whole is homogenated. The tablet cores are coated with the thusobtained mixture in a coating apparatus.

1. A compound of formula (I),

the N-oxide forms, the pharmaceutically acceptable addition salts andthe stereo-chemically isomeric forms thereof, wherein n is 0, 1, 2 or 3and when n is 0 then a direct bond is intended; m is 0, 1, 2 or 3 andwhen m is 0 then a direct bond is intended; t is 0 or 1 and when t is 0then a direct bond is intended; Q is

X is nitrogen; Y is nitrogen; Z is —CH₂— or —O—; R¹ is —C(O)NR³R⁴,—N(H)C(O)R⁷, —C(O)—C₁₋₆alkanediylSR⁷, —NR⁸C(O)N(OH)R⁷,—NR⁸C(O)C₁₋₆alkanediylSR⁷, —NR⁸C(O)C═N(OH)R⁷ or anotherZn-chelating-group wherein R³ and R⁴ are each independently hydrogen,hydroxy, C₁₋₆alkyl, hydroxyC₁₋₆alkyl, aminoC₁₋₆alkyl or aminoaryl; R⁷ ishydrogen, C₁₋₆alkyl, C₁₋₆alkylcarbonyl, arylC₁₋₆alkyl,C₁₋₆alkylpyrazinyl, pyridinone, pyrrolidinone or methylimidazolyl; andR⁸ is hydrogen or C₁₋₆alkyl; R² is hydrogen, hydroxy, amino,hydroxyC₁₋₆alkyl, C₁₋₆alkyl, C₁₋₆alkyloxy, arylC₁₋₆alkyl, aminocarbonyl,hydroxycarbonyl, aminoC₁₋₆alkyl, aminocarbonylC₁₋₆alkyl,hydroxycarbonylC₁₋₆alkyl, hydroxyaminocarbonyl, C₁₋₆alkyloxycarbonyl,C₁₋₆alkylaminoC₁₋₆alkyl or di(C₁₋₆alkyl)aminoC₁₋₆alkyl; -L- is abivalent radical that is C₁₋₆alkanediyl, carbonyl, sulfonyl, orC₁₋₆alkanediyl substituted with phenyl;

is a radical that is

wherein each s is independently 0, 1, 2, 3, 4 or 5; each R⁵ and R⁶ areindependently hydrogen; halo; hydroxy; amino; nitro; trihaloC₁₋₆alkyl;trihaloC₁₋₆alkyloxy; C₁₋₆alkyl; C₁₋₆alkyl substituted with aryl andC₃₋₁₀cycloalkyl; C₁₋₆alkyloxy; C₁₋₆alkyloxyC₁₋₆alkyloxy;C₁₋₆alkylcarbonyl; C₁₋₆alkyloxycarbonyl; C₁₋₆alkylsulfonyl;cyanoC₁₋₆alkyl; hydroxyC₁₋₆alkyl; hydroxyC₁₋₆alkyloxy;hydroxyC₁₋₆alkylamino; aminoC₁₋₆alkyloxy; di(C₁₋₆alkyl)aminocarbonyl;di(hydroxyC₁₋₆alkyl)amino; (aryl)(C₁₋₆alkyl)amino;di(C₁₋₆alkyl)aminoC₁₋₆alkyloxy; di(C₁₋₆alkyl)aminoC₁₋₆alkylamino;di(C₁₋₆alkyl)aminoC₁₋₆alkylaminoC₁₋₆alkyl; arylsulfonyl;arylsulfonylamino; aryloxy; aryloxyC₁₋₆alkyl; arylC₂₋₆alkenediyl;di(C₁₋₆alkyl)amino; di(C₁₋₆alkyl)aminoC₁₋₆alkyl;di(C₁₋₆alkyl)amino(C₁₋₆alkyl)amino;di(C₁₋₆alkyl)amino(C₁₋₆alkyl)aminoC₁₋₆alkyl;di(C₁₋₆alkyl)aminoC₁₋₆alkyl(C₁₋₆alkyl)amino;di(C₁₋₆alkyl)aminoC₁₋₆alkyl(C₁₋₆alkyl)aminoC₁₋₆alkyl;aminosulfonylamino(C₁₋₆alkyl)amino;aminosulfonylamino(C₁₋₆alkyl)aminoC₁₋₆alkyl;di(C₁₋₆alkyl)aminosulfonylamino(C₁₋₆alkyl)amino;di(C₁₋₆alkyl)aminosulfonylamino(C₁₋₆alkyl)aminoC₁₋₆alkyl; cyano;thiophenyl; thiophenyl substituted withdi(C₁₋₆alkyl)aminoC₁₋₆alkyl(C₁₋₆alkyl)aminoC₁₋₆alkyl,di(C₁₋₆alkyl)aminoC₁₋₆alkyl, C₁₋₆alkylpiperazinylC₁₋₆alkyl,hydroxyC₁₋₆alkylpiperazinylC₁₋₆alkyl,hydroxyC₁₋₆alkyloxyC₁₋₆alkylpiperazinylC₁₋₆alkyl,di(C₁₋₆alkyl)aminosulfonylpiperazinylC₁₋₆alkyl, C₁₋₆alkyloxypiperidinyl,C₁₋₆alkyloxypiperidinylC₁₋₆alkyl, morpholinylC₁₋₆alkyl,hydroxyC₁₋₆alkyl(C₁₋₆alkyl)aminoC₁₋₆alkyl, ordi(hydroxyC₁₋₆alkyl)aminoC₁₋₆-alkyl; furanyl; furanyl substituted withhydroxyC₁₋₆alkyl; benzofuranyl; imidazolyl; oxazolyl; oxazolylsubstituted with aryl and C₁₋₆alkyl; C₁₋₆alkyltriazolyl; tetrazolyl;pyrrolidinyl; pyrrolyl; piperidinylC₁₋₆alkyloxy; morpholinyl;C₁₋₆alkylmorpholinyl; morpholinylC₁₋₆alkyloxy; morpholinylC₁₋₆alkyl;morpholinylC₁₋₆alkylamino; morpholinylC₁₋₆alkylaminoC₁₋₆alkyl;piperazinyl; C₁₋₆alkylpiperazinyl; C₁₋₆alkylpiperazinylC₁₋₆alkyloxy;piperazinylC₁₋₆alkyl; naphtalenylsulfonylpiperazinyl;naphtalenylsulfonylpiperidinyl; naphtalenylsulfonyl:C₁₋₆alkylpiperazinylC₁₋₆alkyl; C₁₋₆alkylpiperazinylC₁₋₆alkylamino;C₁₋₆alkylpiperazinylC₁₋₆alkylaminoC₁₋₆alkyl;C₁₋₆alkylpiperazinylsulfonyl; aminosulfonylpiperazinylC₁₋₆alkyloxy;aminosulfonylpiperazinyl; aminosulfonylpiperazinylC₁₋₆alkyl;di(C₁₋₆alkyl)aminosulfonylpiperazinyl;di(C₁₋₆alkyl)aminosulfonylpiperazinylC₁₋₆alkyl;hydroxyC₁₋₆alkylpiperazinyl; hydroxyC₁₋₆alkylpiperazinylC₁₋₆alkyl;C₁₋₆alkyloxypiperidinyl; C₁₋₆alkyloxypiperidinylC₁₋₆alkyl;piperidinylaminoC₁₋₆alkylamino; piperidinylaminoC₁₋₆alkylaminoC₁₋₆alkyl;(C₁₋₆alkylpiperidinyl)(hydroxyC₁₋₆alkyl)aminoC₁₋₆alkylamino;(C₁₋₆alkylpiperidinyl)(hydroxyC₁₋₆alkyl)aminoC₁₋₆alkylaminoC₁₋₆alkyl;hydroxyC₁₋₆alkyloxyC₁₋₆alkylpiperazinyl;hydroxyC₁₋₆alkyloxyC₁₋₆alkylpiperazinylC₁₋₆alkyl;(hydroxyC₁₋₆alkyl)(C₁₋₆alkyl)amino;(hydroxyC₁₋₆alkyl)(C₁₋₆alkyl)aminoC₁₋₆alkyl;hydroxyC₁₋₆alkylaminoC₁₋₆alkyl; di(hydroxyC₁₋₆alkyl)aminoC₁₋₆alkyl;pyrrolidinylC₁₋₆alkyl; pyrrolidinylC₁₋₆alkyloxy; pyrazolyl;thiopyrazolyl; pyrazolyl substituted with two substituents selected fromC₁₋₆alkyl or trihaloC₁₋₆alkyl; pyridinyl; pyridinyl substituted withC₁₋₆alkyloxy, aryloxy or aryl; pyrimidinyl;tetrahydropyrimidinylpiperazinyl;tetrahydropyrimidinylpiperazinylC₁₋₆alkyl; quinolinyl; indolyl; phenyl;phenyl substituted with one, two or three substituents independentlyselected from halo, amino, nitro, C₁₋₆alkyl, C₁₋₆alkyloxy,hydroxyC₁₋₄alkyl, trifluoromethyl, trifluoromethyloxy,hydroxyC₁₋₄alkyloxy, C₁₋₄alkylsulfonyl, C₁₋₄alkyloxyC₁₋₄alkyloxy,C₁₋₄alkyloxycarbonyl, aminoC₁₋₄alkyloxy, di(C₁₋₄alkyl)aminoC₁₋₄alkyloxy,di(C₁₋₄alkyl)amino, di(C₁₋₄alkyl)aminocarbonyl,di(C₁₋₄alkyl)aminoC₁₋₄alkyl, di(C₁₋₄alkyl)aminoC₁₋₄alkylaminoC₁₋₄alkyl,di(C₁₋₄alkyl)amino(C₁₋₄alkyl)amino,di(C₁₋₄alkyl)amino(C₁₋₄alkyl)aminoC₁₋₄alkyl,di(C₁₋₄alkyl)aminoC₁₋₄alkyl(C₁₋₄alkyl)amino,di(C₁₋₄alkyl)aminoC₁₋₄alkyl(C₁₋₄alkyl)aminoC₁₋₄alkyl,aminosulfonylamino(C₁₋₄alkyl)amino,aminosulfonylamino(C₁₋₄alkyl)aminoC₁₋₄alkyl,di(C₁₋₄alkyl)aminosulfonylamino(C₁₋₄alkyl)amino,di(C₁₋₄alkyl)aminosulfonylamino(C₁₋₄alkyl)aminoC₁₋₆alkyl, cyano,piperidinylC₁₋₄alkyloxy, pyrrolidinylC₁₋₄alkyloxy,aminosulfonylpiperazinyl, aminosulfonylpiperazinylC₁₋₄alkyl,di(C₁₋₄alkyl)aminosulfonylpiperazinyl,di(C₁₋₄alkyl)aminosulfonylpiperazinylC₁₋₄alkyl,hydroxyC₁₋₄alkylpiperazinyl, hydroxyC₁₋₄alkylpiperazinylC₁₋₄alkyl,C₁₋₄alkyloxypiperidinyl, C₁₋₄alkyloxypiperidinylC₁₋₄alkyl,hydroxyC₁₋₄alkyloxyC₁₋₄alkylpiperazinyl,hydroxyC₁₋₄alkyloxyC₁₋₄alkylpiperazinylC₁₋₄alkyl,(hydroxyC₁₋₄alkyl)(C₁₋₄alkyl)amino,(hydroxyC₁₋₄alkyl)(C₁₋₄alkyl)aminoC₁₋₄alkyl, di(hydroxyC₁₋₄alkyl)amino,di(hydroxyC₁₋₄alkyl)aminoC₁₋₄alkyl, furanyl, furanyl substituted with—CH═CH—CH═CH—, pyrrolidinylC₁₋₄alkyl, pyrrolidinylC₁₋₄alkyloxy,morpholinyl, morpholinylC₁₋₄alkyloxy, morpholinylC₁₋₄alkyl,morpholinylC₁₋₄alkylamino, morpholinylC₁₋₄alkylaminoC₁₋₄alkyl,piperazinyl, C₁₋₄alkylpiperazinyl, C₁₋₄alkylpiperazinylC₁₋₄alkyloxy,piperazinylC₁₋₄alkyl, C₁₋₄alkylpiperazinylC₁₋₄alkyl,C₁₋₄alkylpiperazinylC₁₋₄alkylamino,C₁₋₄alkylpiperazinylC₁₋₄alkylaminoC₁₋₆alkyl,tetrahydropyrimidinylpiperazinyl,tetrahydropyrimidinylpiperazinylC₁₋₄alkyl,piperidinylaminoC₁₋₄alkylamino, piperidinylaminoC₁₋₄alkylaminoC₁₋₄alkyl,(C₁₋₄alkylpiperidinyl)(hydroxyC₁₋₄alkyl)aminoC₁₋₄alkylamino,(C₁₋₄alkylpiperidinyl)(hydroxyC₁₋₄alkyl)aminoC₁₋₄alkylaminoC₁₋₄alkyl,pyridinylC₁₋₄alkyloxy, hydroxyC₁₋₄alkylamino,hydroxyC₁₋₄alkylaminoC₁₋₄alkyl, di(C₁₋₄alkyl)aminoC₁₋₄alkylamino,aminothiadiazolyl, aminosulfonylpiperazinylC₁₋₄alkyloxy, orthiophenylC₁₋₄alkylamino; each R⁵ and R⁶ can be placed on the nitrogenin replacement of the hydrogen; wherein aryl in the above is phenyl, orphenyl substituted with one or more substituents that is eachindependently halo, C₁₋₆alkyl, C₁₋₆alkyloxy, trifluoromethyl, cyano orhydroxycarbonyl.
 2. A compound as claimed in claim 1 wherein t is 0; R¹is —C(O)NR³R⁴, —C(O)—C₁₋₆alkanediylSR⁷, —NR⁸C(O)N(OH)R⁷,—NR⁸C(O)C₁₋₆alkanediylSR⁷, —NR⁸C(O)C═N(OH)R⁷ or anotherZn-chelating-group wherein R³ and R⁴ are each independently selectedfrom hydrogen, hydroxy, hydroxyC₁₋₆alkyl or aminoC₁₋₆alkyl; R² ishydrogen, hydroxy, amino, hydroxyC₁₋₆alkyl, C₁₋₆alkyl, C₁₋₆alkyloxy,arylC₁₋₆alkyl, aminocarbonyl, aminoC₁₋₆alkyl, C₁₋₆alkylaminoC₁₋₆alkyl ordi(C₁₋₆alkyl)aminoC₁₋₆alkyl; -L- is a bivalent radical selected fromC₁₋₆alkanediyl, carbonyl or sulfonyl;

is a radical selected from (a-1), (a-3), (a-4), (a-5), (a-6), (a-7),(a-8), (a-9), (a-10), (a-11), (a-12), (a-13), (a-14), (a-15), (a-16),(a-17), (a-18), (a-19), (a-20), (a-21), (a-22), (a-23), (a-24), (a-25),(a-26), (a-28), (a-29), (a-30), (a-31), (a-32), (a-33), (a-34), (a-35),(a-36), (a-37), (a-38), (a-39), (a-40), (a-41), (a-42), (a-44), (a-45),(a-46), (a-47), (a-48) or (a-51); s is 0, 1, 2, 3 or 4; R⁵ is hydrogen;halo; hydroxy; amino; nitro; trihaloC₁₋₆alkyl; trihaloC₁₋₆alkyloxy;C₁₋₆alkyl; C₁₋₆alkyloxy; C₁₋₆alkylcarbonyl; C₁₋₆alkyloxyoarbonyl;C₁₋₆alkylsulfonyl; hydroxyC₁₋₆alkyl; aryloxy; di(C₁₋₆alkyl)amino; cyano;thiophenyl; furanyl; furanyl substituted with hydroxyC₁₋₆alkyl;benzofuranyl; imidazolyl; oxazolyl; oxazolyl substituted with aryl andC₁₋₆alkyl; C₁₋₆alkyltriazolyl; tetrazolyl; pyrrolidinyl; pyrrolyl;morpholinyl; C₁₋₆alkylmorpholinyl; piperazinyl; C₁₋₆alkylpiperazinyl;hydroxyC₁₋₆alkylpiperazinyl; C₁₋₆alkyloxypiperidinyl; pyrazoly;pyrazolyl substituted with one or two substituents selected fromC₁₋₆alkyl or trihaloC₁₋₆alkyl; pyridinyl; pyridinyl substituted withC₁₋₆alkyloxy, aryloxy or aryl; pyrimidinyl; quinolinyl; indole; phenyl;or phenyl substituted with one or two substituents independentlyselected from halo, C₁₋₆alkyl, C₁₋₆alkyloxy or trifluoromethyl; and R⁶is hydrogen; halo; hydroxy; amino; nitro; trihaloC₁₋₆alkyl;trihaloC₁₋₆alkyloxy; C₁₋₆alkyl; C₁₋₆alkyloxy; C₁₋₆alkylcarbonyl;C₁₋₆alkyloxyoarbonyl; C₁₋₆alkylsulfonyl; hydroxyC₁₋₆alkyl; aryloxy;di(C₁₋₆alkyl)amino; cyano; pyridinyl; phenyl; or phenyl substituted withone or two substituents independently selected from halo, C₁₋₆alkyl,C₁₋₆alkyloxy or trifluoromethyl.
 3. A compound as claimed in claim 1wherein n is 0, 1 or 2; m is 0, 1 or 2; R¹ is —C(O)NH(OH); R² ishydrogen; -L- is a bivalent radical that is carbonyl,

sulfonyl, or C₁₋₆alkanediyl substituted with phenyl; is a radical thatis (a-1), (a-20) or (a-43); s is 0 or 1; and each R⁵ is hydrogen orphenyl.
 4. A compound as claimed in claim 1 wherein n is 0, 1 or 2; m is1 or 2; R^(al) is —C(O)NH(OH); R² is hydrogen; -L- is a bivalent radicalthat is carbonyl or sulfonyl;

is a radical that is (a-1) or (a-20); each s is independently 0 or 1;and each R⁵ is independently selected from hydrogen or aryl.
 5. Acompound according to claim 1 that is


6. A pharmaceutical composition comprising pharmaceutically acceptablecarriers and as an active ingredient a therapeutically effective amountof a compound as claimed in claim
 1. 7. A process of preparing apharmaceutical composition as claimed in claim 6 wherein thepharmaceutically acceptable carriers and the compound are intimatelymixed.
 8. A method of treating proliferative disease comprisingadministering to a patient in need of such treatment, ananti-proliferative disease-effective amount of a compound of claim
 1. 9.A process for preparing a compound as claimed in claim 1, comprisingreacting an intermediate of formula (II) with an appropriate acid,yielding a hydroxamic acid of formula (I-a), wherein R¹ is —C(O)NH(OH).


10. The method of claim 9 wherein the acid is trifluoroacetic acid. 11.A method of detecting or identifying a histone deactylase (HDAC) in abiological sample comprising detecting or measuring the formation of acomplex between a labelled compound as defined in claim 1 and a HDAC.12. A combination of an anti-cancer agents and a compound of claim 1.